Use of an IL12 receptor-beta 1 splice variant to diagnose active tuberculosis

ABSTRACT

The present invention describes compositions for both diagnostic and therapeutic applications. In one embodiment, the present invention contemplates a method of identifying an active  M. tuberculosis  infection. In another embodiment, the present invention contemplates a method of monitoring a  M. tuberculosis  infection. In yet another embodiment, the present invention contemplates a method of monitoring a patient&#39;s response to treatment for an active  M. tuberculosis  infection. In a further embodiment, the present invention contemplates a method of monitoring a patient&#39;s response to treatment for an active  M. tuberculosis  infection.

This application claims the benefit of priority to Non-Provisionalapplication U.S. Ser. No. 13/022,224, now U.S. Pat. No. 8,394,593, whichwas filed on Feb. 7, 2011, which claims the benefit of ProvisionalApplication U.S. Ser. No. 61/304,025, which was filed on Feb. 12, 2012,the disclosures of which are incorporated herein by reference.

The invention was made with government support under grant number R01AI067723 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates generally to methods and compositions forboth diagnostic and therapeutic applications. In one embodiment, thepresent invention contemplates a method of identifying an active M.tuberculosis infection. In another embodiment, the present inventioncontemplates a method of monitoring a M. tuberculosis infection. In yetanother embodiment, the present invention contemplates a method ofmonitoring a patient's response to treatment for an active M.tuberculosis infection. In a further embodiment, the present inventioncontemplates a method of monitoring a patient's response to treatmentfor an active M. tuberculosis infection.

BACKGROUND

The IL12 Receptor Beta 1 (IL12Rβ1) gene is transcribed in two isoformsby human peripheral blood-derived dendritic cells (HPBDC) when exposedto Mycobacterium tuberculosis (Robinson et al. J. Exp. Med. 2010.207:591-605). Absence of IL-12Rβ1 function predisposes humans toincreased susceptibility to tuberculosis (de Beaucoudrey et al, Medicine2010. 89: 381-402), but no function has been ascribed to the alternativesplice variant (IL12Rβ1 isoform 2), which is induced strongly by M.tuberculosis. Since IL12Rβ1 is known to mediate the activity of thecytokines including IL-12p70, IL-23 and IL-12(p40)₂ it has the potentialto impact many aspects of the immune response. These cytokines areinvolved in the regulation of damaging inflammatory responses associatedwith chronic diseases and are also of interest to researchers studyingprotective immunity to pathogens.

Tuberculosis (TB) is a common, and in many cases lethal, infectiousdisease caused by various strains of mycobacteria. The main cause oftuberculosis is M. tuberculosis, a small, aerobic, nonmotile bacillus.Tuberculosis typically attacks the lungs, but can also affect otherparts of the body. It is spread through the air when people who have anactive tuberculosis infection cough, sneeze, or otherwise transmit theirsaliva through the air. About 90% of those infected have asymptomatic,latent tuberculosis infections, with only a 10% lifetime chance that thelatent infection will progress to overt, active tuberculosis. However,latent infections that progresses to active disease kill more than 50%of individuals if untreated. Diagnosis of active tuberculosis relies onradiology (commonly chest X-rays), as well as microscopic examinationand microbiological culture of body fluids. Diagnosis of latenttuberculosis relies on the tuberculin skin test (TST) and/or bloodtests. Treatment is difficult and requires administration of multipleantibiotics over a long period of time. A definitive diagnosis oftuberculosis is made by identifying M. tuberculosis in a clinical sample(e.g. sputum, pus, or a tissue biopsy). However, the difficult cultureprocess for this slow-growing organism can take two to six weeks forblood or sputum culture.

There is a need to differentiate between people who are infected with M.tuberculosis but not developing disease from those that do have or areon the way to developing disease. It is also important to determine whenpeople are responding to treatment.

SUMMARY OF THE INVENTION

The present invention relates generally to methods and compositions forboth diagnostic and therapeutic applications. In one embodiment, thepresent invention contemplates a method of identifying an active M.tuberculosis infection comprising providing a patient infected with M.tuberculosis, determining the amount of IL12Rβ1 isoform 1 mRNA andIL12Rβ1 isoform 2 mRNA in a cell from the patient, diagnosing thepatient as having an active M. tuberculosis infection if the amount ofIL12Rβ1 isoform 2 mRNA is greater than the amount of IL12Rβ1 isoform 1mRNA and initiating a therapeutic treatment on the patient with activeM. tuberculosis. In some embodiments, the cell is a peripheral bloodmononuclear cell. In other embodiments, the cell is a dendritic cell. Insome embodiments, the amount of IL12Rβ1 isoform 1 mRNA and IL12Rβ1isoform 2 mRNA is determined by PCR. In some embodiments, the PCR isreal-time PCR. In further embodiments, the amount of IL12Rβ1 isoform 1mRNA and IL12Rβ1 isoform 2 mRNA is determined by microarraytranscriptional analysis.

In one embodiment, the present invention contemplates a method ofidentifying an active M. tuberculosis infection comprising providing apatient suspected of being infected with M. tuberculosis, determiningthe amount of IL12Rβ1 isoform 1 mRNA and IL12Rβ1 isoform 2 mRNA in acell from the patient, diagnosing the patient as having an active M.tuberculosis infection if the amount of IL12Rβ1 isoform 2 mRNA isgreater than the amount of IL12Rβ1 isoform 1 mRNA and initiating atherapeutic treatment on the patient with active M. tuberculosis.

In one embodiment, the present invention contemplates a method ofmonitoring a M. tuberculosis infection comprising providing a patientinfected with M. tuberculosis, determining the amount of IL12Rβ1 isoform1 mRNA and IL12Rβ1 isoform 2 mRNA in a cell from the patient, diagnosingthe patient as having an active M. tuberculosis infection if the amountof IL12Rβ1 isoform 2 mRNA is greater than the amount of IL12Rβ1 isoform1 mRNA, diagnosing the patient as having a latent M. tuberculosisinfection if the amount of IL12Rβ1 isoform 1 mRNA is greater than theamount of IL12Rβ1 isoform 2 mRNA and initiating a therapeutic treatmentif the patient has an active M. tuberculosis infection. In oneembodiment, the cell is a peripheral blood mononuclear cell. In anotherembodiment, the cell is a dendritic cell. In one embodiment, the amountof IL12Rβ1 isoform 1 mRNA and IL12Rβ1 isoform 2 mRNA is determined byPCR. In yet another embodiment, the PCR is real-time PCR. In a furtherembodiment, the amount of IL12Rβ1 isoform 1 mRNA and IL12Rβ1 isoform 2mRNA is determined by microarray transcriptional analysis.

In one embodiment, the present invention contemplates a method ofmonitoring a patient's response to treatment for an active M.tuberculosis infection, comprising providing a patient with an active M.tuberculosis infection, initiating a therapeutic treatment on thepatient, determining the amount of IL12Rβ1 isoform 1 mRNA and IL12Rβ1isoform 2 mRNA in a cell from the patient and continuing the therapeutictreatment if the amount of IL12Rβ1 isoform 1 mRNA is greater than theamount of IL12Rβ1 isoform 2 mRNA. In one embodiment, the therapeutictreatment is determined to be effective if the amount of IL12Rβ1 isoform2 mRNA decreases below the amount of IL12Rβ1 isoform 1 mRNA. In oneembodiment, the cell is a peripheral blood mononuclear cell. In anotherembodiment, the cell is a dendritic cell. In one embodiment, the amountof IL12Rβ1 isoform 1mRNA and IL12Rβ1 isoform 2 mRNA is determined byPCR. In another embodiment, the PCR is real-time PCR. In yet anotherembodiment, the amount of IL12Rβ1 isoform 1 mRNA and IL12Rβ1 isoform 2mRNA is determined by microarray transcriptional analysis.

In one embodiment, the present invention contemplates a method ofmonitoring a patient's response to treatment for an active M.tuberculosis infection comprising, providing a patient with an active M.tuberculosis infection, initiating a therapeutic treatment on thepatient, determining the amount of IL12Rβ1 isoform 1 mRNA and IL12Rβ1isoform 2 mRNA in a cell from the patient during the course of thetreatment and diagnosing the patient as responding to the treatment whenthe amount of IL12Rβ1 isoform 2 mRNA is less than the amount of IL12Rβ1isoform 1 mRNA. In one embodiment the cell is a peripheral bloodmononuclear cell. In another embodiment the cell is a dendritic cell. Inone embodiment, the amount of IL12Rβ1 isoform 1mRNA and IL12Rβ1 isoform2 mRNA is determined by PCR. In another embodiment, the PCR is real-timePCR. In yet another embodiment, the amount of IL12Rβ1 isoform 1 mRNA andIL12Rβ1 isoform 2 mRNA is determined by microarray transcriptionalanalysis.

In one embodiment, the present invention contemplates a vaccineformulation comprising an antigen and the IL12Rβ1 isoform 2. In someembodiments this invention relates to a method of quantifying the ratioof IL12Rβ1 cDNA and a splice variant thereof in a sample. In otherembodiments, this invention relates to a method of augmenting an immuneresponse by administering, inhibiting and/or inducing the IL12Rβ1isoform 2.

In some embodiments, the present invention relates generally to avaccine formulation comprising an antigen and the IL12Rβ1 isoform 2.

In some embodiments, the present invention relates generally to a methodfor quantifying a transcript and a splice variant of said transcript fordiagnostic purposes. In one embodiment, the method comprises providing asample that comprises cDNA molecules encoding IL12Rβ1 isoform 1 andIL12Rβ1 isoform 2, a PCR primer set flanking the transmembrane-encodingregion of the cDNA molecules, and a fluorescent-conjugated primer,amplifying the cDNAs with the PCR primer set, labeling the products ofthe PCR amplification with the fluorescent-conjugated primer anddetecting the labeled PCR products. In some embodiments, the nucleotidesequence of the forward PCR primer is SEQ ID NO: 1. In some embodiments,the nucleotide sequence of the reverse PCR primer is SEQ ID NO:2. Inother embodiments, detecting the labeled PCR products further comprisesdetecting the ratio of transcript encoding IL12Rβ1 isoform 1 to splicevariant encoding IL12Rβ1 isoform 2. In further embodiments, the sampleis isolated from a cell. In still further embodiments, the cell is adendritic cell. In some embodiments, the cell has been exposed to apathogen. In other embodiments, the pathogen is Mycobacteriumtuberculosis.

In some embodiments, the present invention relates generally to a methodof augmenting an immune response comprising, providing a subject and apeptide isoform of an IL12Rβ1 splice variant, and administering saidpeptide isoform to said subject. In some embodiments, the peptideisoform is the IL12Rβ1 isoform 2. In some embodiments, the peptideisoform is a fragment of the IL12Rβ1 isoform 2. In other embodiments,administering the splice variant enhances a type-1 cellular immuneresponse in the subject. In other embodiments, the splice variant isadministered concomitant with a vaccination. In still other embodiments,the splice variant is administered concomitant with an immunotherapy. Inother embodiments, at least a fragment of said peptide isoform isadministered to said subject.

In some embodiments, the present invention relates generally to a methodof augmenting an immune response, comprising providing a subject and aninhibitor of a peptide isoform of an IL12Rβ1 splice variant, andadministering the inhibitor to the subject. In some embodiments, thepeptide isoform is IL12Rβ1 isoform 2. In some embodiments, the peptideisoform is a fragment of the IL12Rβ1 isoform 2. In other embodiments,the splice variant is administered concomitant with a vaccination. Inother embodiments, the inhibitor comprises a monoclonal or polyclonalantibody specific for the IL12Rβ1 isoform 2. In further embodiments, theinhibitor comprises a siRNA molecule specific for the splice variantencoding the IL12Rβ1 isoform 2. In still further embodiments,administering the inhibitor limits a type-1 cellular immune response inthe subject. In some embodiments, administering the inhibitor limits aninflammatory immune response in said subject. In other embodiments, theinflammatory response is an IL12 dominated immune response.

In some embodiments, the present invention relates generally to a methodof augmenting an immune response comprising, providing a subject and acompound capable of inducing expression of a peptide isoform of anIL12Rβ1 splice variant, and administering the compound to the subjectsuch that expression of the peptide isoform is induced. In someembodiments, the peptide isoform is the IL12Rβ1 isoform 2. In someembodiments, the peptide isoform is a fragment of the IL12Rβ1 isoform 2.In some embodiments, the compound is a subunit of Mycobacteriumtuberculosis. In some embodiments, the compound is a glycolipid moleculeof Mycobacterium tuberculosis. In other embodiments, inducing expressionof the splice variant enhances a type-1 cellular immune response in thesubject. In other embodiments, the splice variant is induced concomitantwith a vaccination. In still other embodiments, the splice variant isinduced concomitant with an immunotherapy.

In some embodiments, the present invention relates generally to a primerhaving the nucleotide sequence of SEQ ID NO: 1.(5′-ACACTCTGGGTGGAATCCTG-3′ [Forward])

In some embodiments, the present invention relates generally to a primerset comprising a first primer having the nucleotide sequence of SEQ IDNO: 1 and a second primer having the nucleotide sequence of SEQ ID NO:2. (5′GCCAACTTGGACACCTTGAT-3′ [Reverse])

In some embodiments, the present invention relates generally to a kitcomprising a primer set comprising a first primer having the nucleotidesequence of SEQ ID NO: 1 and a second primer having the nucleotidesequence of SEQ ID NO: 2.

In some embodiments, the present invention relates generally to avaccine formulation comprising an antigen and a peptide isoform of thesplice variant IL12Rβ1ΔTM.

In some embodiments, the present invention relates generally to a methodfor quantifying a transcript and a splice variant of said transcript fordiagnostic purposes. In one embodiment, the method comprises providing asample that comprises IL12Rβ1 and IL12Rβ1ΔTM cDNA molecules, a PCRprimer set flanking the transmembrane-encoding region of the cDNAmolecules, and a fluorescent-conjugated primer, amplifying the cDNAswith the PCR primer set, labeling the products of the PCR amplificationwith the fluorescent-conjugated primer and detecting the labeled PCRproducts. In some embodiments, detecting the labeled PCR productsfurther comprises detecting the ratio of IL12Rβ1 to IL12Rβ1 ΔTM. Infurther embodiments, the sample is isolated from a cell. In stillfurther embodiments, the cell is a dendritic cell. In some embodiments,the cell has been exposed to a pathogen. In other embodiments, thepathogen is Mycobacterium tuberculosis.

In some embodiments, the present invention relates generally to a methodof augmenting an immune response comprising, providing a subject and apeptide isoform of an IL12Rβ1 splice variant, and administering saidpeptide isoform to said subject. In some embodiments, the peptideisoform is the IL12Rβ1 splice variant IL12Rβ1ΔTM. In some embodiments,the peptide isoform is a fragment of the IL12Rβ1 splice variantIL12Rβ1ΔTM. In other embodiments, administering the splice variantenhances a type-1 cellular immune response in the subject. In otherembodiments, the splice variant is administered concomitant with avaccination. In still other embodiments, the splice variant isadministered concomitant with an immunotherapy. In other embodiments, afragment of said peptide isoform is administered to said subject.

In some embodiments, the present invention relates generally to a methodof augmenting an immune response, comprising providing a subject and aninhibitor of a peptide isoform of an IL12Rβ1 splice variant, andadministering the inhibitor to the subject. In some embodiments, thepeptide isoform is the IL12Rβ1 splice variant IL12Rβ1ΔTM. In someembodiments, the peptide isoform is a fragment of the IL12Rβ1 splicevariant IL12Rβ1ΔTM. In other embodiments, the splice variant isadministered concomitant with a vaccination. In other embodiments, theinhibitor comprises a monoclonal or polyclonal antibody specific for thepeptide isoform IL12Rβ1ΔTM. In further embodiments, the inhibitorcomprises a siRNA molecule specific for the mRNA encoding the peptideisoform IL12Rβ1ΔTM. In still further embodiments, administering theinhibitor limits a type-1 cellular immune response in the subject. Insome embodiments, administering the inhibitor limits an inflammatoryimmune response in said subject. In other embodiments, the inflammatoryresponse is an IL12 dominated immune response.

In some embodiments, the present invention relates generally to a methodof augmenting an immune response comprising, providing a subject and acompound capable of inducing expression of a peptide isoform of anIL12Rβ1 splice variant, and administering the compound to the subjectsuch that expression of the peptide isoform is induced. In someembodiments, the peptide isoform is the splice variant IL12Rβ1ΔTM. Insome embodiments, the peptide isoform is a fragment of the IL12Rβ1splice variant IL12Rβ1ΔTM. In some embodiments, the compound is asubunit of Mycobacterium tuberculosis. In some embodiments, the compoundis a glycolipid molecule of Mycobacterium tuberculosis. In otherembodiments, inducing expression of the splice variant enhances a type-1cellular immune response in the subject. In other embodiments, thesplice variant is induced concomitant with a vaccination. In still otherembodiments, the splice variant is induced concomitant with animmunotherapy.

DEFINITIONS

To facilitate the understanding of this invention a number of terms aredefined below. Terms defined herein (unless otherwise specified) havemeanings as commonly understood by a person of ordinary skill in theareas relevant to the present invention. Terms such as “a”, “an” and“the” are not intended to refer to only a singular entity, but includethe general class of which a specific example may be used forillustration. The terminology herein is used to describe specificembodiments of the invention, but their usage does not delimit theinvention, except as outlined in the claims.

As used herein, terms defined in the singular are intended to includethose terms defined in the plural and vice versa.

As used herein, the terms “patient” and “subject” refer to a human oranimal who is ill or who is undergoing treatment for disease, but doesnot necessarily need to be hospitalized. For example, out-patients,persons in nursing homes are “patients”.

As used herein, the term “concomitant” refers to existing, occurring oraccompanying together or along with something else, sometimes (but notalways) in a lesser way (e.g. a peptide of IL12Rβ1 isoform 2 may beadministered concomitant with a vaccine formulation such that theimmunizing effects of the vaccine are enhanced).

As used herein, the term “nucleic acid” refers to a covalently linkedsequence of nucleotides in which the 3′ position of the pentose of onenucleotide is joined by a phosphodiester group to the 5′ position of thepentose of the next, and in which the nucleotide residues (bases) arelinked in specific sequence; i.e., a linear order of nucleotides. A“polynucleotide”, as used herein, is a nucleic acid containing asequence that is greater than about 100 nucleotides in length. Nucleicacid molecules are said to have a “5′-terminus” (5′ end) and a“3′-terminus” (3′ end) because nucleic acid phosphodiester linkagesoccur to the 5′ carbon and 3′ carbon of the pentose ring of thesubstituent mononucleotides. The end of a nucleic acid at which a newlinkage would be to a 5′ pentose carbon is its 5′ terminal nucleotide(by convention sequences are written, from right to left, in the 5′ to3′ direction). The end of a nucleic acid at which a new linkage would beto a 3′ pentose carbon is its 3′ terminal nucleotide. A terminalnucleotide, as used herein, is the nucleotide at the end position of the3′- or 5′-terminus. DNA molecules are said to have “5′ ends” and “3′ends” because mononucleotides are reacted to make oligonucleotides in amanner such that the 5′ phosphate of one mononucleotide pentose ring isattached to the 3′ oxygen of its neighbor in one direction via aphosphodiester linkage. Therefore, an end of an oligonucleotide isreferred to as the “5′ end” if its 5′ phosphate is not linked to the 3′oxygen of a mononucleotide pentose ring and as the “3′ end” if its 3′oxygen is not linked to a 5′ phosphate of a subsequent mononucleotidepentose ring.

As used herein, the term “oligonucleotide” refers to a shortpolynucleotide or a portion of a polynucleotide comprised of two or moredeoxyribonucleotides or ribonucleotides, preferably more than three, andusually more than ten. The exact size will depend on many factors, whichin turn depends on the ultimate function or use of the oligonucleotide.The oligonucleotide may be generated in any manner, including chemicalsynthesis, DNA replication, reverse transcription, or a combinationthereof. The word “oligo” is sometimes used in place of the word“oligonucleotide”.

As used herein, the term “an oligonucleotide having a nucleotidesequence encoding a gene” or “a nucleic acid sequence encoding” aspecified polypeptide refers to a nucleic acid sequence comprising thecoding region of a gene or in other words the nucleic acid sequence thatencodes a gene product. The coding region may be present in cDNA,genomic DNA or RNA form. When present in a DNA form, the oligonucleotidemay be single-stranded (in other words, the sense strand) ordouble-stranded. Suitable control elements such as enhancers/promoters,splice junctions, polyadenylation signals, etc. may be placed in closeproximity to the coding region of the gene if needed to permit properinitiation of transcription and/or correct processing of the primary RNAtranscript. Alternatively, the coding region utilized in the expressionvectors of the present invention may contain endogenousenhancers/promoters, splice junctions, intervening sequences,polyadenylation signals, etc. or a combination of both endogenous andexogenous control elements.

As used herein, the term “gene” refers to a nucleic acid (for example,DNA or RNA) sequence that comprises coding sequences necessary for theproduction of RNA, or a polypeptide or its precursor. A functionalpolypeptide can be encoded by a full-length coding sequence or by anyportion of the coding sequence as long as the desired activity orfunctional properties (for example, enzymatic activity, ligand binding,signal transduction, etc.) of the polypeptide are retained. The term“portion” when used in reference to a gene refers to fragments of thatgene. The fragments may range in size from a few nucleotides to theentire gene sequence minus one nucleotide. Thus, “a nucleotidecomprising at least a portion of a gene” may comprise fragments of thegene or the entire gene. The term “gene” also encompasses the codingregions of a structural gene and includes sequences located adjacent tothe coding region on both the 5′ and 3′ ends for a distance of about 1kb on either end such that the gene corresponds to the length of thefull-length mRNA. The sequences which are located 5′ of the codingregion and which are present on the mRNA are referred to as 5′non-translated sequences. The sequences which are located 3′ ordownstream of the coding region and which are present on the mRNA arereferred to as 3′ non-translated sequences. The term “gene” encompassesboth cDNA and genomic forms of a gene. A genomic form or clone of a genecontains the coding region interrupted with non-coding sequences termed“introns” or “intervening regions” or “intervening sequences”. Intronsare segments of a gene that are transcribed into nuclear RNA (hnRNA);introns may contain regulatory elements such as enhancers. Introns areremoved or “spliced out” from the nuclear or primary transcript; intronstherefore are absent in the messenger RNA (mRNA) transcript. The mRNAfunctions during translation to specify the sequence or order of aminoacids in a nascent polypeptide.

As used herein, the term “coding region” refers to the nucleotidesequences that encode the amino acids found in the nascent polypeptideas a result of translation of an mRNA molecule. The coding region isbounded, in eukaryotes, on the 5′ side by the nucleotide triplet “ATG”which encodes the initiator methionine and on the 3′ side by one of thethree triplets that specify stop codons (i.e., TAA, TAG, TGA).

As used herein, the term the terms “peptide”, “peptide sequence”, “aminoacid sequence”, “polypeptide”, and “polypeptide sequence” are usedinterchangeably herein to refer to at least two amino acids or aminoacid analogs that are covalently linked by a peptide bond or an analogof a peptide bond. The term peptide includes oligomers and polymers ofamino acids or amino acid analogs. The term peptide also includesmolecules that are commonly referred to as peptides, which generallycontain from about two (2) to about twenty (20) amino acids. The termpeptide also includes molecules that are commonly referred to aspolypeptides, which generally contain from about twenty (20) to aboutfifty amino acids (50). The term peptide also includes molecules thatare commonly referred to as proteins, which generally contain from aboutfifty (50) to about three thousand (3000) amino acids. The amino acidsof the peptide may be L-amino acids or D-amino acids. A peptide,polypeptide or protein may be synthetic, recombinant or naturallyoccurring. A synthetic peptide is a peptide that is produced byartificial means in vitro.

As used herein, the term “alternative splicing” refers to the process bywhich the exons of the RNA produced by transcription of a gene (aprimary gene transcript or pre-mRNA) are reconnected in multiple waysduring RNA splicing. The resulting different mRNAs, referred to as“splice variants” or “alternative splice variants”, may be translatedinto different protein isoforms; thus a single gene may code formultiple proteins. In eukaryotes, alternative splicing greatly increasesthe diversity of proteins that can be encoded by the genome. In humans,for example, over 80% of genes are alternatively spliced. There arenumerous modes of alternative splicing, such as exon skipping in which aparticular exon may be included in an mRNA under certain conditions (orin certain tissues) and omitted from the mRNA under other conditions.For example, IL12Rβ1ΔTM mRNA is an alternative splice variant of theIL-12 Receptor Beta 1 (IL12Rβ1) gene involved in IL-12 signalingpathways. Using PCR primer sets that flank (i.e. hybridize to regions 3′and 5′) an alternative splice site (i.e. splice junction) it is possibleto amplify cDNA molecules representing both the spliced and unsplicedRNA molecules. PCR amplification products produced from the spliced cDNAtemplate will be smaller than those produced from the unspliced cDNAtemplate.

As used herein, the term “polymerase chain reaction” (“PCR”) refers tothe method of Mullis as provided for in U.S. Pat. Nos. 4,683,195,4,683,202, and 4,965,188, incorporated herein by reference, thatdescribe a method for increasing the concentration of a segment of atarget sequence in a mixture of genomic DNA without cloning orpurification. This process for amplifying the target sequence consistsof introducing a large excess of two oligonucleotide primers to the DNAmixture containing the desired target sequence, followed by a precisesequence of thermal cycling in the presence of a DNA polymerase. The twoprimers are complementary to their respective strands of the doublestranded target sequence. To effect amplification, the mixture isdenatured and the primers then annealed to their complementary sequenceswithin the target molecule. Following annealing, the primers areextended with a polymerase so as to form a new pair of complementarystrands. The steps of denaturation, primer annealing, and polymeraseextension can be repeated many times (in other words, denaturation,annealing and extension constitute one “cycle”; there can be numerous“cycles”) to obtain a high concentration of an amplified segment of thedesired target sequence. The length of the amplified segment of thedesired target sequence is determined by the relative positions of theprimers with respect to each other, and therefore, this length is acontrollable parameter. By virtue of the repeating aspect of theprocess, the method is referred to as the “polymerase chain reaction”(hereinafter “PCR”). Because the desired amplified segments of thetarget sequence become the predominant sequences (in terms ofconcentration) in the mixture, they are said to be “PCR amplified”. WithPCR, it is possible to amplify a single copy of a specific targetsequence in genomic DNA to a level detectable by several differentmethodologies (for example, hybridization with a labeled probe;incorporation of biotinylated primers followed by avidin-enzymeconjugate detection; incorporation of ³²P-labeled deoxynucleotidetriphosphates, such as dCTP or dATP, into the amplified segment). Inaddition to genomic DNA, any oligonucleotide or polynucleotide sequencecan be amplified with the appropriate set of primer molecules. Inparticular, the amplified segments created by the PCR process itselfare, themselves, efficient templates for subsequent PCR amplifications.

As used herein, the terms “PCR product”, “PCR fragment” and“amplification product” refer to the resultant mixture of compoundsafter two or more cycles of the PCR steps of denaturation, annealing andextension are complete. These terms encompass the case where there hasbeen amplification of one or more segments of one or more targetsequences.

As used herein, the term “primer” refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, which is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product which is complementary to a nucleic acid strand isinduced, (in other words, in the presence of nucleotides and an inducingagent such as DNA polymerase and at a suitable temperature and pH). Theprimer is preferably single stranded for maximum efficiency inamplification, but may alternatively be double stranded. If doublestranded, the primer is first treated to separate its strands beforebeing used to prepare extension products. Preferably, the primer is anoligodeoxyribonucleotide. The primer must be sufficiently long to primethe synthesis of extension products in the presence of the inducingagent. The exact lengths of the primers will depend on many factors,including temperature, source of primer and the use of the method.

As used herein, the term “real time PCR” or “Taqman real time PCR”refers to a modified PCR that allows simultaneous amplification andquantification of a specific target DNA or cDNA molecule usingsequence-specific RNA or DNA-based reporter probes. The reported probeonly hybridizes to DNA or cDNA targets that contain the probe sequence,thereby significantly increasing specificity and allowing quantificationeven in the presence of non-specific amplification. The reported probetypically bears a fluorescent reporter at one end of the DNA or RNAmolecule and a quencher of that fluorescence at the opposite end of themolecule. The quencher molecule blocks the fluorescence emitted by thefluorophore when excited by the PCR cycler's light source via FRET(Fluorescence Resonance Energy Transfer). As long as the fluorophore andthe quencher are in proximity, quenching inhibits any fluorescencesignals. As the Taq polymerase extends the primer and synthesizes thenascent strand, the 5′ to 3′ exonuclease activity of the Taq polymerasedegrades the probe that has annealed to the template. Degradation of theprobe releases the fluorophore such that it is no longer in closeproximity to the quencher, thus relieving the quenching effect andallowing fluorescence of the fluorophore. Fluorescence detected in thereal-time PCR thermal cycler is therefore directly proportional to thefluorophore released and the amount of DNA template present in the PCR.The product targeted by the reporter probe at each PCR cycle thereforecauses a proportional increase in fluorescence due to the breakdown ofthe probe and release of the reporter. TaqMan probes may, for example,consist of a fluorophore covalently attached to the 5′-end of anoligonucleotide probe and a quencher at its 3′-end. Several differentfluorophores are available, such as 6-carboxyfluorescein (i.e. FAM) ortetrachlorofluorescin (i.e. TET). Likewise, several different quenchersare also available, such as tetramethylrhodamine (i.e. TAMRA) ordihydrocyclopyrroloindole tripeptide minor groove binder (i.e. MGB).This potentially allows for multiplex assays for several genes in thesame reaction by using specific probes with different colored labels,provided that all genes are amplified with similar efficiency. In someembodiments a real time PCR reaction may be performed by using anon-specific fluorescent dye (including for example SYBR green) thatbind with the double-stranded DNA generated during the PCR reactionrather than the above-described probe bearing a fluorescent reporter atone end of the a quencher molecule at the other end.

As used herein, an “aerosol” is defined as a suspension of liquid orsolid particles of a substance (or substances) in a gas. The presentinvention contemplates the use of both atomizers and nebulizers ofvarious types. An “atomizer” is an aerosol generator without a baffle,whereas a “nebulizer” uses a baffle to produce smaller particles.

As used herein, the term “shRNA” or “short hairpin RNA” refers to asequence of ribonucleotides comprising a single-stranded RNA polymerthat makes a tight hairpin turn on itself to provide a “double-stranded”or duplexed region. shRNA can be used to silence gene expression via RNAinterference. shRNA hairpin is cleaved into short interfering RNAs(siRNA) by the cellular machinery and then bound to the RNA-inducedsilencing complex (RISC). It is believed that the complex inhibits RNA,completely or partially, as a consequence of the complexed siRNAhybridizing to and cleaving RNAs that match the siRNA that is boundthereto.

As used herein, the term “RNA interference” or “RNAi” refers to thesilencing or decreasing of gene expression by siRNAs. It is the processof sequence-specific, post-transcriptional gene silencing in animals andplants, initiated by siRNA that is homologous in its duplex region tothe sequence of the silenced gene. The gene may be endogenous orexogenous to the organism, present integrated into a chromosome orpresent in a transfection vector that is not integrated into the genome.The expression of the gene is either completely or partially inhibited.RNAi inhibits the gene by compromising the function of a target RNA,completely or partially. Both plants and animals mediate RNAi by theRNA-induced silencing complex (RISC); a sequence-specific,multicomponent nuclease that destroys messenger RNAs homologous to thesilencing trigger. RISC is known to contain short RNAs (approximately 22nucleotides) derived from the double-stranded RNA trigger, although theprotein components of this activity are unknown. However, the22-nucleotide RNA sequences are homologous to the target gene that isbeing suppressed. Thus, the 22-nucleotide sequences appear to serve asguide sequences to instruct a multicomponent nuclease, RISC, to destroythe specific mRNAs. Carthew has reported (Curr. Opin. Cell Biol. 13(2):244-248 (2001)) that eukaryotes silence gene expression in the presenceof dsRNA homologous to the silenced gene. Biochemical reactions thatrecapitulate this phenomenon generate RNA fragments of 21 to 23nucleotides from the double-stranded RNA. These stably associate with anRNA endonuclease, and probably serve as a discriminator to select mRNAs.Once selected, mRNAs are cleaved at sites 21 to 23 nucleotides apart.

As used herein, the term “siRNAs” refers to short interfering RNAs. Insome embodiments, siRNAs comprise a duplex, or double-stranded region,of about 18-25 nucleotides long; often siRNAs contain from about two tofour unpaired nucleotides at the 3′ end of each strand. At least onestrand of the duplex or double-stranded region of a siRNA issubstantially homologous to or substantially complementary to a targetRNA molecule. The strand complementary to a target RNA molecule is the“antisense strand”; the strand homologous to the target RNA molecule isthe “sense strand”, and is also complementary to the siRNA antisensestrand. siRNAs may also contain additional sequences; non-limitingexamples of such sequences include (but are not limited to) linkingsequences, or loops, as well as stem and other folded structures. siRNAsappear to function as key intermediaries in triggering RNA interferencein invertebrates and in vertebrates, and in triggering sequence-specificRNA degradation during posttranscriptional gene silencing in plants.

As used herein, the term “antibody” or “antibodies” refers to globularproteins (“immunoglobulins”) produced by cells of the immune system toidentify and neutralize foreign antigens. “Monoclonal antibodies” (mAb)are antibodies that recognize a specific antigenic epitope (i.e.monospecific) because they are derived from clones of a singlehybridoma. Hybridomas are cells engineered to produce a desired mAbantibody in large amounts. Briefly, B-cells are removed from the spleenof an animal that has been challenged with the desired antigen. TheseB-cells are then fused with myeloma tumor cells that can growindefinitely (i.e. immortal) in culture. Since the fused cell orhybridoma is also immortal it will multiply rapidly and indefinitely toproduce large amounts of the desired mAb (Winter and Milstein, Nature,349, 293-299, 1991). “Polyclonal antibodies” (pAb) are a mixture ofantibodies that recognize multiple epitopes of a specific antigen.Polyclonal antibodies are produced by immunizing an animal (i.e. mouse,rabbit, goat, horse, sheep etc.) with a desired antigen to induceB-lymphocytes to produce antibodies to multiple epitopes of thatantigen. These antibodies can then be isolated from the animal's bloodusing well-known methods, such as column chromatography.

As used herein, the term “lymphocyte” refers to white blood cells thatinclude B lymphocytes (B cells) and T lymphocytes (T cells). IndividualB cells and T cells specifically recognize a single antigenic epitopeand also recognize the body's own (self) tissues as different fromnon-self tissues. After B cells and T cells are formed, a smallpopulation will multiply and provide “memory” for the immune system.This allows the immune system to respond faster and more efficiently thenext time you are exposed to the same antigen.

As used herein, the terms “inhibit”, “inhibition”, “inhibitor” or“suppress” and grammatical equivalents thereof, refer to the act ofdiminishing, suppressing, alleviating, limiting, eliminating,preventing, blocking and/or decreasing an action and/or function; as forexample the inhibition of a chemical reaction or biological process. Asused herein, it is not necessary that there be complete inhibition, itis sufficient for there to be some inhibition. For example, a compoundthat inhibits cancer may kill all cancerous cells or prevent, arrest orslow further cancerous cell growth. These terms find use in both invitro as well as in vivo systems.

As used herein, the terms “reduce” and “reduction” and grammaticalequivalents thereof, means lowering, decreasing, or diminishing indegree, intensity, extent, and/or amount. As used herein, it is notnecessary that there be complete reduction, it is sufficient for thereto be some reduction.

As used herein, the terms “prevent” and “preventing” and grammaticalequivalents thereof, indicates the hindrance of the recurrence, spreador onset of a disease or disorder. It is not intended that the presentinvention be limited to complete prevention. In some embodiments, theonset is delayed, or the severity of the disease or disorder is reduced.

As used herein, the terms “treat”, “treating”, “treatment” andgrammatical equivalents thereof, refers to combating a disease ordisorder, as for example in the management and care of a patient.“Treatment” is not limited to cases where the subject (e.g. patient) iscured and the disease is eradicated. Rather, the present invention alsocontemplates treatment that merely reduces symptoms, improves (to somedegree) and/or delays disease progression. It is not intended that thepresent invention be limited to instances wherein a disease oraffliction is cured. It is sufficient that symptoms are reduced.

As used herein, the term “downregulate” or “downregulation” refers to adecrease, relative to an appropriate control, in the amount of a givenmolecule, protein, gene product, or nucleic acid such as DNA or RNA dueto exposure to or contact with an inhibitor.

As used herein, the terms “diagnose” “diagnosis” or “diagnosing” refersto the recognition of a disease by its signs and symptoms (e.g.,resistance to conventional therapies), or genetic analysis, pathologicalanalysis, histological analysis, and the like.

As used herein, a “diagnostic” is a compound or method that assists inthe identification and characterization of a health or disease state.With regard to the present invention, it is contemplated that a methodfor determining the ratio of cDNA molecules encoding IL12Rβ1 isoform 1to cDNA molecules encoding the splice variant IL12Rβ1 isoform 2 can beused as a diagnostic to evaluate the course of an immune response in apatient following an infection. For example, a patient infected with M.tuberculosis may be examined with such a diagnostic to determine whethera particular cytokine response has been stimulated as well as therelative levels of such cytokines.

As used herein, the term “cytokines” refers to a category of protein,peptide, or glycoprotein molecules secreted by specific cells of theimmune system that carry signals between cells. Cytokines are a criticalcomponent of both the innate and adaptive immune response, and are oftensecreted by immune cells that have encountered a pathogen to activateand recruit additional immune cells to increase the system's response tothe pathogen. Cytokines are typically released in the general region ofthe pathogen-infected cells such that responding immune cells arrive atthat site of infection. Each individual cytokine has a matchingcell-surface receptor. Upon binding of a cytokine to its cell-surfacereceptor a cascade of intracellular signaling events alters the cell'sfunction. This includes the upregulation and/or downregulation of genesinvolved in the production of other cytokines, an increase expression ofsurface receptors for other molecules, or suppression of the cytokineitself by feedback inhibition. The effect of a particular cytokine on agiven cell depends on the cytokine, its extracellular abundance, thepresence and abundance of the complementary receptor on the cellsurface, and downstream signals activated by receptor binding. Commoncytokines include interleukins that are responsible for communicationbetween white blood cells; chemokines that promote chemotaxis; andinterferons that have anti-viral effects, such as shutting down proteinsynthesis in the host cell. Cytokines are characterized by considerable“redundancy”, in that many cytokines appear to share similar functions.

Interleukin 12 (IL-12), also known as natural killer cell stimulatoryfactor (NKSF), T cell stimulatory factor, or cytotoxic lymphocytematuration factor (CLMF), is a cytokine produced by dendritic cells(DCs), macrophages and B-cells in response to antigenic stimulation.IL-12 plays a central role in the initiation and regulation of cellularimmune responses, including the differentiation of naive T cells intoeither Th1 or Th2 cells; a crucial in determining the type of reactionelicited in response to a particular pathogen. In addition to enhancingthe cytotoxic activity of natural killer (NK) cells and CD8⁺ cytotoxic Tcells, IL-12 also stimulates the production of the cytokinesinterferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) by Tand natural killer (NK) cells. IL-12 also has anti-angiogenic activity,which means it can block the formation of new blood vessels. It doesthis by increasing production of interferon gamma, which in turnincreases the production of the inducible protein-10 (IP-10) chemokineIP-10 then mediates this anti-angiogenic effect.

IL-12 binds to the heterodimeric IL-12 receptor (CD212) formed byIL12Rβ1 and IL12Rβ2 subunits. IL12Rβ2 plays a central role in IL-12function, since it is found on activated T cells and is stimulated bycytokines that promote Th1 cell development and inhibited by those thatpromote Th2 cell development. Upon binding, IL12Rβ2 becomes tyrosinephosphorylated and provides binding sites for the Tyk2 and Jak2 kinasesof the JAK-STAT pathway. These kinases are important in activatingtranscription factors (such as STAT4) involved in IL-12 signaling in Tcells and NK cells. IL12 receptors are present on activated CD4⁺ andCD8⁺ positive T-cells and activated NK cells. IL-2 stimulates expressionof the IL-12 receptors (IL12Rβ1 and IL12Rβ2), critical receptor proteinsinvolved in IL-12 signaling in NK cells.

As used herein, the term “chemotaxis” or “chemotactic” refers to themovement or orientation of an organism or cell along a chemicalconcentration gradient either toward or away from the chemical stimulus.Movement towards a chemical stimulus is referred to as “positivechemotaxis”, while movement away from a chemical stimulus is referred toas “negative chemotaxis”. Chemotaxis requires cell motility (the abilityto move spontaneously and independently), a specific receptor torecognize the chemical stimulus and a signaling pathway linking thereceptor to the element(s) controlling the movement. Chemotaxis occursin both single-cell and multi-cellular organisms. For example, bacteriaexhibit chemotaxis when they move toward a source of nutrients (such asglucose) or move away from a poison (such as phenol). Multicellularorganisms also utilize chemotaxis for numerous aspects of theirdevelopment, including for example, the movement of sperm towards theegg during fertilization and the migration of neurons. A variety ofimmune cells (including granulocytes, monocytes and lymphocytes) areattracted to the site of infection by the release of chemotacticcytokines known as chemokines NK cells, CD4⁺ and CD8⁺ T cells andpolymorphonuclear cells (PMNs) have all been demonstrated to exhibit apositive chemotaxis response to IL-12 (Blood, 84(7): 2261-2268). Inaddition, subversion of the normal chemotaxis mechanism is a recognizedfactor in cancer metastasis.

As used herein, the term “T helper cell”, “effector T cell” or “Th cell”refers to a sub-group of T lymphocytes involved in establishing andmaximizing the capabilities of the immune system. While Th cells lackcytotoxic or phagocytic activity, they activate and direct other immunecells, such as B-cell antibody class switching and the activation andgrowth of cytotoxic T cells. Th cells are also involved in maximizingthe activity of phagocytes such as macrophages. Mature Th cells expressthe surface protein CD4, and are therefore referred to as CD4⁺ T cells.Th cells differentiate into two major subtypes of cells known as Type 1(Th-1) and Type 2 (Th-2) helper cells, respectively.

As used herein, the term “cell-mediated immunity” refers to an immuneresponse that does not involve antibodies or complement but ratherinvolves the activation of macrophages, natural killer cells (NK),antigen-specific cytotoxic T-lymphocytes (T-cells), and the release ofvarious cytokines in response to an antigen. Patterns of cytokineproduction by T cells are associated with different immunologicalresponses, described as type-1 (Th-1) and type-2 (Th-2) responses. Insome embodiments, the Th-1 response stimulates cell-mediated immunity byreleasing cytokines such as IFN-γ, which increase the production ofIL-12 by dendritic cells and macrophages. In some embodiments, 11-12also stimulates the production of IFN-γ in Th-1 cells by positivefeedback. In further embodiments, IFN-γ also inhibits the production ofcytokines associated with the Th-2 response, such as interleukin-4,thereby preserving the Th-1 response. In some embodiments, the Th-2response stimulates the humoral immune system by promoting theproliferation of antibody producing B-cells. In some embodiments, theTh-2 response involves the release of cytokines such as IL-4 thatfurther promotes the production of Th-2 cytokines. In other embodiments,the Th-2 response releases IL-10, which inhibits the production of Th-1related cytokines such as interleukin-2 and IFN-γ in T helper cells andIL-12 in dendritic cells and macrophages.

As used herein, the term “inflammatory response” refers to inflammationthat occurs when tissues are injured by any number of causes, includingfor example, bacteria or virus infections, trauma, toxins and/or heat.Chemicals released by the damaged tissues (including cytokines,histamine, bradykinin and serotonin) cause blood vessels to leak fluidinto the surrounding tissues resulting in local swelling. This helpsisolate the foreign substance from further contact with body tissues.These chemicals also attract immune cells that function to clearmicroorganisms and dead or damaged cells by the process of phagocytosis.

As used herein, the term “dendritic cell” or “DC” refers to immune cellsthat form part of the mammalian immune system. The main function of DCsis to functioning as “antigen-presenting cells” by processing foreignantigens and presenting antigenic epitopes on their surface to othercells of the immune system. DCs are present in small quantities intissues that are in contact with the external environment, mainly theskin (where there is a specialized dendritic cell type called Langerhanscells) and the inner lining of the nose, lungs, stomach and intestines.DCs can also be found in an immature state in the blood. Once activated,they migrate to the lymphoid tissues where they interact with T cellsand B cells to initiate the adaptive immune response. At certaindevelopment stages DCs grow branched projections (dendrites) that givethe cell its name. In some embodiments, DCs can be differentiated intotwo sub-populations based on the expression of the cell surface markerCD11c. In some embodiments, CD11c⁺ DCs produce IL12 and stimulate a Th1response in lymphocytes, while CD11c⁻ DCs synthesize little IL12 but area major source of alpha-interferon and stimulate lymphocytes to produceTh2 cytokines.

As used herein, the term “vaccine”, “vaccinate” or “vaccination” refersto the introduction of a small amount of an antigen into an organism inorder to trigger an immune system that generates activated B cellsand/or sensitized T cells. These cells recognize and eliminate theforeign antigen and also establish immune system “memory” such thatfuture exposures to the antigen result in its rapid recognition andclearance. A variety of antigenic substances may be used forvaccination, including dead or inactivated (i.e. live attenuated)organisms or purified products derived therefrom. Vaccines can be usedto prevent or ameliorate the effects of a future infection (i.e.prophylactic) or therapeutic, such as anti-cancer vaccine.

As used herein, the term “immunotherapy” refers to the treatment of adisease by inducing, enhancing or suppressing an immune response.Immunotherapies designed to elicit or amplify an immune response areclassified as “activation immunotherapies”, while those designed toreduce, suppress or direct an existing immune response are classified as“suppression immunotherapies”. Immunotherapy agents may include adiverse array of recombinant, synthetic and natural preparations,including cytokines for example.

As used herein, the term “ELISPOT assay” or “Enzyme-Linked ImmunosorbentSpot Assay” refers to a method for monitoring immune responses in humansand animals developed by Cecil Czerkinsky. The ELISPOT assay is amodified version of the ELISA immunoassay and was originally developedto enumerate B cells secreting antigen-specific antibodies. This assayhas subsequently been adapted for various tasks, including theidentification and enumeration of cytokine-producing cells at the singlecell level. Briefly, the ELISPOT assay permits visualization of thesecretory product of individual activated or responding cells. Each“spot” that develops in the assay represents a single reactive cell.Thus, the ELISPOT assay provides both qualitative (type of immuneprotein) and quantitative (number of responding cells) information. Thesensitivity of the ELISPOT assay permits frequency analysis of rare cellpopulations (e.g., antigen-specific responses). This sensitivity is duein part to the ability to rapidly capture the product around thesecreting cell before it is diluted in the supernatant, captured byreceptors of adjacent cells, or degraded. This makes ELISPOT assays muchmore sensitive than conventional ELISA measurements. Limits of detectionare below 1/100,000 rendering the assay uniquely useful for monitoringantigen-specific responses, applicable to a wide range of areas ofimmunology research, including cancer, transplantation, infectiousdisease, and vaccine development.

As used herein, the term “Mycobacterium tuberculosis” refers to apathogenic bacterial species in the genus Mycobacterium that isprimarily a pathogen of mammalian respiratory systems and is thecausative agent of most cases of tuberculosis. The cell surface of M.tuberculosis has a waxy coating composed primarily of mycolic acid,which renders the cell impervious to Gram staining. Not all individualsinfected with M. tuberculosis bacteria become sick. As a result, two M.tuberculosis-related conditions exist: latent M. tuberculosis infectionand M. tuberculosis disease (i.e. active M. tuberculosis). Both latentM. tuberculosis infection and active M. tuberculosis can be treated.Individuals with latent M. tuberculosis infections have M. tuberculosisbacteria in their bodies, but they do not become sick because thebacteria are not active. Latently infected individuals are asymptomaticand cannot spread M. tuberculosis bacteria to others. However, if the M.tuberculosis bacteria become active in and multiply, the latentlyinfected individual may proceed from having a latent M. tuberculosisinfection to having active M. tuberculosis disease. For this reason,people with a latent M. tuberculosis infection are often prescribedtreatment to prevent them from developing active M. tuberculosisdisease. Treatment of latent M. tuberculosis infection is essential forcontrolling and eliminating M. tuberculosis in the United States. Theoverall goal for treatment of M. tuberculosis infections is not only tocure the individual patient but also to minimize the transmission ofMycobacterium tuberculosis to other persons. Thus, successful treatmentof M. tuberculosis has benefits both for the individual patient and thecommunity in which the patient resides. In some embodiments, anindividual with a latent M. tuberculosis infection may need to take justone type of anti-tuberculosis medication. Individuals with active M.tuberculosis infections, particularly those individuals infected with adrug-resistant strain of M. tuberculosis, may require several drugs atonce. Common medications used to treat tuberculosis include (but are notnecessarily limited to) Isoniazid, Rifampin (Rifadin, Rimactane),Ethambutol (Myambutol) and Pyrazinamide. Regimens for treating M.tuberculosis disease typically have an initial phase of 2 months,followed by a choice of several options for the continuation phase ofeither 4 or 7 months, resulting in a total treatment regimen of 6 to 9months. It is very important that individuals infected with M.tuberculosis complete the entire therapeutic regimen exactly asprescribed. Ceasing the therapeutic regimen too soon may result in theinfected individual becoming sick again. Failure to follow theprescribed regimen correctly may result in the remaining M. tuberculosisbacteria that are still alive to become resistant to those drugs. M.tuberculosis bacteria that have established drug resistance are muchmore difficult and expensive to treat. Completion of the therapeuticregimen is determined by the number of doses of drug ingested over agiven period of time. Although basic M. tuberculosis regimens arebroadly applicable, there are modifications that should be made underspecial circumstances (such as people with HIV infection, drugresistance, pregnancy, or treatment of children).

As used herein, the term “therapeutic” refers to drugs and the method oftheir administration in the treatment of disease, including for exampleM. tuberculosis. Similarly, the term “drug” refers generally to avariety of molecules (including biological and/or chemical molecules) orpharmacological compounds that prevent, treat, or reduce at least onesymptom of a disease such as M. tuberculosis. Examples of “therapeutics”used to treat tuberculosis include (but are in no way limited to)Isoniazid, Rifampin (Rifadin, Rimactane), Ethambutol (Myambutol) andPyrazinamide.

As used herein, the term “fluorescence” refers to the emission ofvisible light by a substance that has absorbed light of a differentwavelength. In some embodiments, fluorescence provides a non-destructivemeans of tracking and/or analyzing biological molecules based on thefluorescent emission at a specific frequency. Proteins (includingantibodies), peptides, nucleic acid, oligonucleotides (including singlestranded and double stranded primers) may be “labeled” with a variety ofextrinsic fluorescent molecules referred to as fluorophores.Isothiocyanate derivatives of fluorescein, such as carboxyfluorescein,are an example of fluorophores that may be conjugated to proteins (suchas antibodies for immunohistochemistry) or nucleic acids. In someembodiments, fluorescein may be conjugated to nucleoside triphosphatesand incorporated into nucleic acid probes (such as“fluorescent-conjugated primers”) for in situ hybridization. In someembodiments, a molecule that is conjugated to carboxyfluorescein isreferred to as “FAM-labeled”.

As used herein, the term “array” or “microarray” refers to a multiplextechnology for high-throughput screening of large amounts of biologicalmaterial (including protein, RNA or DNA) consisting of an arrayed seriesof molecules on a solid substrate, such as a glass slide or thin-filmsilicon cell. Types of microarrays include DNA microarrays (includingcDNA microarrays, oligonucleotide microarrays and single nucleotidepolymorphisms (SNP) microarrays), MMChips (i.e. for surveillance ofmicroRNA populations), protein microarrays, tissue microarrays, cellularmicroarrays (i.e. transfection microarrays), chemical compoundmicroarrays, antibody microarrays and carbohydrate arrays (i.e.glycoarrays). A DNA microarray consists of an arrayed series ofthousands of microscopic spots of DNA oligonucleotides (i.e. features)containing picomoles (10⁻¹² moles) of a specific DNA sequence (i.e.probes or reporters). These may be a short section of a gene or otherDNA element that are used to hybridize a cDNA sample (i.e. target) underhigh-stringency conditions. Probe-target hybridization may be detectedand quantified using, for example, fluorophore-, silver-, orchemiluminescence-labeled targets to determine relative abundance ofnucleic acid sequences in the target. Since a microarray may containtens of thousands of probes, a single experiment may accomplish manygenetic tests in parallel to dramatically accelerate data generation andanalysis. In standard microarrays, the probes are attached via surfaceengineering to a solid surface by a covalent bond to a chemical matrix(i.e. via epoxy-silane, amino-silane, lysine, polyacrylamide or others).The solid surface can be glass or a silicon chip, typically referred toas a “gene chip” or “Affymetrix chip”. Other microarray platforms, suchas Illumina, use microscopic beads, instead of the large solid support.DNA arrays are different from other types of microarray only in thatthey either measure DNA or use DNA as part of the detection system. DNAmicroarrays may be used to measure changes in expression levels(including for example “microarray transcriptional analysis”), to detectSNPs and to genotype or re-sequence genomes. Microarrays also differ infabrication, function, accuracy, efficiency and cost. In someembodiments, the terms “array” and “microarray” are usedinterchangeably, differing only in general size. In one embodiment, thepresent invention contemplates the same methods for making and usingeither. An array typically contains many cells (typically100-1,000,000⁺) wherein each cell is at a known location and contains aspecific component of interest. Each array therefore contains numerousdifferent components of interest. In some embodiments, arrays may beused to validate the clinical relevance of potential biological targetsin the development of diagnostics, therapeutics and to study new diseasemarkers and genes. Tissue arrays are suitable for genomics-baseddiagnostic and drug target discovery.

BRIEF DESCRIPTION OF THE FIGURES

For a more complete understanding of the features and advantages of thepresent invention, reference is now made to the detailed description ofthe invention along with the accompanying figures.

FIG. 1 depicts the role of IL12Rβ1 in M. tuberculosis-induced DCmigration. (A) M. tuberculosis was instilled into the trachea of C57BL/6mice and 3 hrs later the frequency of CD11c⁺ IL12Rβ1⁺ cells in the lungswas determined. Dot plots are representative of four mice per condition;this experiment was performed twice. (B) C57BL/6 bone marrow-derived DCs(BMDCs) were exposed to M. tuberculosis or media alone and 3 hrs laterthe frequency of CD11c⁺ IL12Rβ1⁺ cells was determined. Dot plotsrepresent the same BMDC preparation stimulated with either condition andare representative of three separate experiments. (C) BMDCs generatedfrom C57BL/6 or IL12Rβ1^(−/−) mice (where IL12Rβ1^(−/−) indicates anabsence of both IL12Rβ1 alleles as compared to IL12Rβ1^(+/+) whichindicates that both IL12Rβ1 alleles are present) were assayed for theirability to migrate to CCL19 in a transwell assay after a 3 hr exposureto M. tuberculosis chemotaxis index (CI) represents the number moved inresponse to CCL19/number moved to media alone. Data points in (C)represent mean and standard deviation (SD) of triplicate values and arerepresentative of three separate experiments; for the difference betweenCI induced in C57BL/6 relative to IL12Rβ1^(−/−) DCs, *p<0.05, **p<0.005as determined by Student's t-test. (D-F) M. tuberculosis/CFSE wasinstilled via the trachea into C57BL/6, il12b^(−/−), IL12Rβ1^(−/−) oril12rb2^(−/−) mice. 18 hrs later the frequency (D,E) and total number(F) of CD11c⁺CFSE⁺ cells in the draining MLN were counted. The datapoints (E,F) represent the mean and SD of combined data from 4 mice pergroup and are representative of two separate experiments; for thedifference between percentage and/or number of CD11c⁺CFSE⁺ cells foundin C57BL/6 mice relative to il12b^(−/−) or IL12Rβ1^(−/−) mice, *p<0.05,***p<0.0005 as determined by Student's t-test.

FIG. 2 demonstrates that the presence of IL12Rβ1^(−/−) DCs in the lungassociates with impaired activation of M. tuberculosis-specific T cellsin the draining MLN. Chimeras comprising 75% Itgax-DTR/EGFP:25%IL12Rβ1^(+/+) or 75% Itgax-DTR/EGFP:25% IL12Rβ1^(−/−) were injected witheither PBS (A,F) or DT (B-E, G-J). 12 hrs later the frequency of CD11c⁺GFP⁺ and CD11c⁺ GFP⁻ cells remaining in the lungs after PBS injection(A,F) or DT injection (B,G) was determined. Gating based on CD11c⁺ GFP⁺or CD11c⁺ GFP⁻ cells demonstrated the level of IL12Rβ1 surfaceexpression (A,F). DT injected mice subsequently received 1.5×10⁶CFSE-labeled ESAT₆-specific CD4⁺ T cells and 1 μg ESAT6₁₋₂₀/50 ngirradiated M. tuberculosis via the trachea. 12 hrs later the frequencyof CFSE⁺CD4⁺ cells in the draining MLN (C, H) and expression levels ofthe activation markers CD69 (D, I) and CD44 (E, J) were determined byflow cytometry. The data points (K, L) represent the CD44 (K) and CD69(L) data from 5 mice per group that received either 1 μg or 10 ngESAT₁₋₂₀ peptide with irradiated M. tuberculosis and are representativeof two separate experiments; for the difference in % CD44^(hi) and %CD69⁺ ESAT-specific CD4⁺ cells between the indicated groups, *p<0.05,**p<0.005 as determined by Student's t-test.

FIG. 3 demonstrates that NFκB signaling is impaired in IL12Rβ1^(−/−) DCsand can be promoted by IL12(p40)₂. BMDCs generated from C57BL/6 andIL12Rβ1^(−/−) mice were exposed to M. tuberculosis (B,D) or media alone(A,C) for the indicated times. Cells were then harvested undernon-denaturing conditions and levels of total NFκB p65 (open bars) andphospho-NFκB p65 (closed bars) were determined by ELISA. Shown are theabsorbance values (A₄₅₀) from one experiment that is representative ofthree. (E) BMDCs were generated from il12b^(−/−) mice and exposed tomedia alone, M. tuberculosis, IL12(p40)₂, or both M. tuberculosis andIL12(p40)₂. 1 hr later nuclear extracts of the treated cells wereisolated and electromobility shift analysis (EMSA) of NFκB consensussequence-binding proteins was performed. Shown is a blot of NFκBconsensus sequence-binding proteins from DCs stimulated with media alone(lanes 1,2), M. tuberculosis (lanes 3,4), both M. tuberculosis andIL12(p40)₂ (lanes 5,6) or IL12(p40)₂ alone (lanes 7,8). The absence (−)or presence (+) of a cold NFκB consensus probe was used to determine thespecificity of each band.

FIG. 4 demonstrates that DCs express an IL12Rβ1 mRNA alternative splicevariant following exposure to M. tuberculosis. (A) The genomic positionand organization of the murine IL12Rβ1 locus. Exons 1-16 are denotede.1-e.16. Upon transcription and intron-removal (B) e.1-13 becomes theextracellular-encoding (EC) portion of the IL12Rβ1 transcript, e.14 thetransmembrane-encoding (TM) portion, and e15-16 theintracellular-encoding (IC) portion. Shown to the right of thetranscript are the relative positions of primers 1-5 (P1-P5; ∇ indicatesa forward primer and Δ indicates a reverse primer) used for (C)amplification of IL12Rβ1 cDNA from the indicated cell populations. (D-E)Sequencing of the smaller amplicon of P3-P5 (indicated by arrow) revealsan IL12Rβ1 mRNA alternative splice variant that contains both a 97 bpdeletion and (E) a frameshift insertion that eventually produces apremature stop codon.

FIG. 5 illustrates the steps involved in IL12Rβ1 Spectratype analysis.(A) cDNA is first amplified with primers that flank thetransmembrane-encoding region in order to amplify both IL12Rβ1 andIL12Rβ1ΔTM; the resultant amplicons are then fluorescently (FAM)-labeledvia a run-off PCR reaction with a single FAM-conjugated primer. Giventhe published sequence of IL12Rβ1 and IL12Rβ1ΔTM (Chua et al., JImmunology 155:4286-4299, 1995), the FAM-labeled amplicons of thesetranscripts have a predicted size of 229 by and 132 bp, respectively.(B) Analyzing the samples by fluorescent capillary electrophoresisallows the FAM-labeled products to be separated by size and theirrelative abundance to one another quantified. To demonstrate this, twopeaks of the anticipated sizes are observed using cDNA of concanavalin-Aactivated splenocytes; neither are observed in no-reverse-transcriptasecontrols, ruling out genomic DNA amplification (C). Using the area underthe larger, transmembrane containing fluorescent peak as a unitreference, the relative abundance of IL12Rβ1ΔTM can be determined. Thenumbers adjacent to peaks of individual IL12Rβ1 spectra indicate therelative ratio of that peak's area (the smaller peak representingIL12Rβ1ΔTM) to the area of the larger peak that represents IL12Rβ1. Inconcanavalin A-activated splenocytes, the ratio of IL12Rβ1ΔTM to IL12Rβ1was observed to be 0.4:1 (B). To further test the fidelity of this assayto distinguish between IL12Rβ1 and IL12Rβ1ΔTM, NIH/3T3 cells weretransfected with mammalian expression vectors containing each respectivecDNA. IL12Rβ1 Spectratype analysis of single-(D, E) anddouble-transfectants (F) revealed that the 229 bp and 132 bp peaksobserved via this assay do in fact represent IL12Rβ1 and IL12Rβ1ΔTM,respectively. Importantly, western blot analysis with polyclonalanti-IL12Rβ1 confirmed that IL12Rβ1ΔTM could be translated into aprotein product as first demonstrated by Chua et al. (J Immunology155:4286-4299, 1995) (G). Subcellular fractionation of cell membrane andcell cytosol confirmed IL12Rβ1ΔTM to be membrane associated as firstpredicted by Chua et al. (Chua et al., J Immunology 155:4286-4299, 1995)(H).

FIG. 6 depicts the L12Rβ1 Spectratype analysis of M.tuberculosis-activated DCs. (A) C57BL/6 bone marrow-derived DCs werestimulated over a period of 6 hrs with media alone or M. tuberculosis.Shown are representative IL12Rβ1 spectratype data from these DCs beforeculture (0 hr) and after 1.5, 3 or 6 hrs of culture. The numbersadjacent to peaks of individual IL12Rβ1 spectra indicate the relativeratio of that peak's area (the smaller peak representing IL12Rβ1ΔTM) tothe area of the larger peak that represents IL12Rβ1. Spectra arerepresentative of four mice per condition; this experiment was performedtwice. (B) Denaturing western analysis of the same cells to confirmchanging protein levels of IL12Rβ1 and IL12Rβ1ΔTM; recombinant IL12Rβ1and NIH/3T3 cells transfected with the indicated plasmid constructsserved as positive controls; blots were probed with polyclonalanti-IL12Rβ1.

FIG. 7 depicts IL12Rβ1ΔTM expression by BMDCs following exposure to M.avium, M. avium cell wall extract, Y. pestis, LPS, TNFα, IL12 orIL12(p40)₂. DCs prepared from C57BL/6 bone marrow were exposed in vitroto either media alone, Y. pestis (5 MOI), M. avium (5 MOI), M. aviumcell wall extract, E. coli LPS or to cytokines TNFα, IL12 and IL12(p40)₂for 3 hrs. At the end of 1.5 and 3 hr periods DC RNA was collected forIL12Rβ1 Spectratype analysis. (A) Measurement of IL12p40 in the DCsupernatant by ELISA served as a positive control that both Y. pestisand M. avium were capable of stimulating DCs. (B-H) RepresentativeIL12Rβ1 spectra from 1.5 hr and 3 hr following exposure to (B) M. avium,(C) M. avium cell wall extract, (D) Y. pestis, (E) LPS, (F) TNFα, (G)IL12 or (H) IL12(p40)₂. (I) Western Blot demonstrating that IL12Rβ1ΔTMpeptide production is not observed after stimulation of DCs with varyingMOI of Y. pestis.

FIG. 8 depicts the expression of two IL12Rβ1 isoforms by human DCsfollowing exposure to M. tuberculosis and other specific stimuli. (A-B)Two isoforms of the human IL12Rβ1 transcript are reported in publiclyavailable databases: full length IL12Rβ1 (isoform 1; Swiss-Prot IDP42701-1) and a shorter isoform that is the product of alternativesplicing (isoform 2; Swiss-Prot P42701-3). The amino acid sequences of(A) isoform 1 (SEQ ID NO: 24) and (B) isoform 2 (SEQ ID NO: 25) arereproduced. (C-D) Monocyte-derived DCs were generated by incubatingmagnetically purified CD14⁺ monocytes from apheresis samples for sevendays with GMCSF and IL4. (C) DCs were then incubated for three days witheither media alone, IL1β, IL10, IL2, IL6, PLGF1, CCL3 or for 24 hourswith LPS. (D) Alternatively, DCs were stimulated with M. tuberculosisover a 6 hr period. Subsequently generated cDNA from both (C-D) was thenamplified with primer pairs that either amplified both isoforms 1 and 2(Common), only isoform 1 (isoform 1 specific) or only isoform 2 (isoform2 specific). cDNA from CD3⁺ peripheral blood mononuclear cells (PBMCs)was used as a positive control for IL12Rβ1 expression.

FIG. 9 depicts IL12Rβ1ΔTM expression in M. tuberculosis-infected lungs.C57BL/6 mice were aerogenically infected with 100 colony forming units(CFU) M. tuberculosis. At the indicated times post-infection the lungsof both (A) uninfected and (B) infected mice were harvested for IL12Rβ1Spectratype analysis. Shown are representative spectra from (A) oneindividual uninfected mouse at each indicated time point or (B) twoindividual M. tuberculosis-infected mice from each time point. Thenumbers adjacent to peaks of an individual IL12Rβ1 spectra indicate therelative ratio of that peak's area (the smaller peak representingIL12Rβ1ΔTM) to the area of the larger peak that represents IL12Rβ1. (C)The ratio of IL12Rβ1ΔTM to IL12Rβ1 expressed in the lung of uninfectedand M. tuberculosis-infected animals. (D) The ratio of IL12Rβ1ΔTM toIL12Rβ1 expressed in the liver of uninfected and M.tuberculosis-infected animals. Data points in (C-D) represent the meanand SD of the IL12Rβ1ΔTM to IL12Rβ1 ratios expressed in 4-8 individualmice per time point; for the difference between infected lungs relativeto uninfected lungs, *p<0.05, **p<0.005 as determined by Student'st-test.

FIG. 10 depicts IL12Rβ1ΔTM expression in CD11c⁺ and CD11c⁻ populationsfollowing M. tuberculosis-infection. C57BL/6 mice were aerogenicallyinfected with 100 CFU of M. tuberculosis. At the indicated times afterinfection, lung CD11c⁺ and CD11c⁻ populations were magneticallyseparated from both (A) uninfected and (B) infected mice. Subsequentlygenerated cDNA was used for IL12Rβ1 Spectratype analysis. Representativespectra expressed by CD11c⁺ and CD11c⁻ cells from (A) an individualuninfected mouse at each time point or (B) an individual M.tuberculosis-infected mouse at each time point are shown. The numbersadjacent to peaks of an individual IL12Rβ1 spectrum indicate therelative ratio of that peak's area (the smaller peak representingIL12Rβ1ΔTM) to the area of the larger peak that represents IL12Rβ1.Spectra are representative of four mice per time point. (C) The ratio ofIL12Rβ1ΔTM to IL12Rβ1 expressed by lung CD11c⁺ cells from uninfected andM. tuberculosis-infected animals. (D) The ratio of IL12Rβ1ΔTM to IL12Rβ1expressed by lung CD11c⁻ cells from uninfected and M.tuberculosis-infected animals. Data points in (C-D) represent the meanand SD of the IL12Rβ1ΔTM to IL12Rβ1 ratios expressed in 4 individualmice per time point; for the difference between the indicatedpopulations from infected lungs relative to uninfected lungs, *p<0.05 asdetermined by Student's t-test.

FIG. 11 depicts IL12Rβ1ΔTM expression in the lung following infectionwith M. avium or Y. pestis. C57BL/6 mice were aerogenically infectedwith 1×10³ CFU of M. avium strain 2447. Shown in (A) are the M. aviumCFU per lung at various times post-infection. (B) On days 1, 8, 14 and29 post-infection total lung RNA was harvested for IL12Rβ1 Spectratypeanalysis. Shown are representative spectra from three individual M.avium-infected mice at each time point, with the smaller peakrepresenting IL12Rβ1ΔTM and the larger peak representing IL12Rβ1. (C-D)C57BL/6 were intranasally infected with 1×10⁵ CFU of Y. pestis strainKIMD27 or 1×10⁶ CFU of Y. pestis strain KIMD27 pLpxL. Four dayspost-infection the lungs were harvested to both (C) determine the totalCFU per lung and (D) assess total lung expression of IL12Rβ1ΔTM. Shownare the spectra from five individual Y. pestis KIMD27 or KIMD27 pLpxLinfected mice at this time. Data points in (A, C) represent the meannumber and SD of bacterial CFU present in the lungs of 4-5 individualmice per time point.

FIG. 12 demonstrates that IL12Rβ1ΔTM enhances IL12Rβ1-dependentmigration. (A-D) IL12Rβ1^(−/−) CD11c⁺ BMDCs were transfected with mRNAsencoding either GFP, GFP and IL12Rβ1, GFP and IL12Rβ1ΔTM or GFP andIL12Rβ1 and IL12Rβ1ΔTM. 24 hrs later (A) cells were analyzed by flowcytometry for GFP expression among CD11c⁺ cells and (B) expression oftransfected IL12Rβ1 was examined by gating on GFP⁺CD11c⁺ cells. (C) Themigratory ability of DCs transfected with the indicated mRNAs wasassessed as performed in FIG. 1C. Data points represent the mean and SDof the combined data from three separate experiments. For the differencebetween CI induced in the indicated groups, *p<0.05 as determined byStudent's t-test. (D) Flow cytometric analysis of those cells that hadmigrated and transfected with GFP and IL12Rβ1 and IL12Rβ1βTMdemonstrates that the migratory DCs from this group were mostly GFP⁺.(E) The ability of IL12Rβ1^(−/−) DCs transfected with indicated mRNAs toactivate M. tuberculosis-specific T cells in vivo was compared;sham-transfected C57BL/6 DCs were used as a positive control. Followingtransfection the indicated DCs populations were cultured with M.tuberculosis and ESAT₁₋₂₀ peptide; and then instilled via the tracheainto C57BL/6 mice containing transferred CFSE-labeled ESAT-specific CD4⁺cells. Shown are histograms of CD44 and CD69 expression on CFSE⁺CD4⁺ 12hrs later in the draining MLN. Each histogram is representative of fourmice per condition. (F, G) The combined (F) CD44 and (G) CD69 data gatedon CFSE⁺CD4⁺ in the draining MLN are shown; these data arerepresentative of two independent experiments.

FIG. 13 depicts the role of IL12Rβ1βTM on IL12(p40)₂-dependent NIH/3T3cell migration and STAT4 phosphorylation. (A) NIH/3T3 cells transfectedwith IL12Rβ1 alone, IL12Rβ1βTM alone, both IL12Rβ1βTM and IL12Rβ1 orempty vector were placed in the upper well of a Boyden chamber, whilethe bottom well contained either IL12(p40)₂ or media alone. (B) NIH/3T3cells were transiently transfected with plasmids that constitutivelyexpress IL12Rβ1, IL12Rβ12 and STAT4. Following the addition of IL12,STAT4 phosphorylation is measured using a STAT4-reporter plasmid thatcontains firefly-luciferase under control of the GAS-promoter.

FIG. 14 demonstrates that the inability to generate IL12Rβ1 isoform 2results in reduced ability to limit M. tuberculosis growth in thespleen. Mice lacking the expression of the IL12Rβ1 isoform 2 in eitherCD11c or CD4 expressing cells were infected via the aerosol route with100 CFU of M. tuberculosis and the number of CFU per spleen wasdetermined by viable plating after 15 and 33 days. Knock-in mice are themice with the loxP-flanked (floxed) allele but without any cre andprovide the control for the effect of cre-mediated deletion of theIL12Rβ1 isoform 2. One of two similar experiments is shown. Significancedetermined by ANOVA.

FIG. 15 demonstrates that IL12Rβ1 isoform 2 is expressed at higherlevels in patients with active tuberculosis. RNA was extracted fromhuman peripheral blood and the transcriptional profile determined usingthe Affymetrix Microarray. Samples were grouped based on exposure level(healthy contacts, exposed but not diseased—latent TB, and activedisease—TB) or on period of drug treatment—0-6 months. Data was analyzedfor significance by ANOVA.

FIG. 16 demonstrates that upon exposure to M. tuberculosis humandendritic cells express the IL12Rβ1 isoform 2 in excess relative to theIL12Rβ1 isoform 1. Human monocyte-derived dendritic cells were culturedwith live M. tuberculosis and the mRNA extracted at various times postinfection. The absolute number of copies for each isoform was determinedby PCR and the ratio of the specific mRNAs calculated. The data shownare the means and SD for all 7 unrelated donors tested.

FIG. 17 depicts the primer sequences for the detection of the IL12Rβ1isoform 1 (12-F and 12/13-R) and IL12Rβ1 isoform 2 (9/9b-F and 9b-R).Nucleotide sequence of each primer are as follows: isoform 1 forwardprimer 12-F 5′-ACGTCTCGGTGAAGAATCATA-3′ [SEQ ID NO: 17]; isoform 1reverse primer 12/13-R 5′-GATGCTCTGACACCTGTTT-3′ [SEQ ID NO: 18];isoform 2 forward primer 9/9b-F 5′-ACACCCACACAGATGGCATGA-3′ [SEQ ID NO:19] and isoform 2 reverse primer 9b-R 5′-TTAGCCGGGTATGGTGGCAGAT-3′ [SEQID NO: 20].

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates generally to methods and compositions forboth diagnostic and therapeutic applications. In one embodiment, thepresent invention contemplates a method of identifying an active M.tuberculosis infection. In another embodiment, the present inventioncontemplates a method of monitoring a M. tuberculosis infection. In yetanother embodiment, the present invention contemplates a method ofmonitoring a patient's response to treatment for an active M.tuberculosis infection. In a further embodiment, the present inventioncontemplates a method of monitoring a patient's response to treatmentfor an active M. tuberculosis infection.

I. Dendritic Cells

DCs are pivotal for initiating immunity to M. tuberculosis (Khader etal., J Exp Med, 203:1805-1815, 2006; Tian et al., J Immunol,175:3268-3272, 2005) and other diseases of the pulmonary tract(Lambrecht et al., Curr Mol Med, 8:393-400, 2008). The majority ofindividuals infected with M. tuberculosis control the infection throughan acquired antigen-specific CD4⁺ T-cell response (Mogues et al., J ExpMed, 193:271-280, 2001). The IL12 family of cytokines (i.e. IL12, IL23and IL12(p40)₂) are essential to the generation of this response (Cooperet al., Annu Rev Immunol, 27:393-422, 2009), with IL12(p40)₂ beingrequired for DCs to migrate following mycobacterial and other pathogenicstimuli (Khader et al., J Exp Med, 203:1805-1815, 2006; McCormick etal., J Immunol, 181:2356-2367, 2008; Robinson et al., J Immunol,181:5560-5567, 2008). IL12 cytokine family members are also secreted byDCs following pathogen stimulation (Jang et al., J Leukoc Biol,84:1264-1270, 2008; Robinson et al., J Immunol, 181:5560-5567, 2008) andare required for their ability to generate an efficient T cell response(Robinson et al., J Immunol, 181:5560-5567, 2008; Zhang et al., JImmunol, 171:4485-4492, 2003). After encountering M. tuberculosis,CD11c⁺ DCs migrate from the lung to the draining mediastinal lymph node(MLN) where they present M. tuberculosis antigen(s) to T cells (Wolf etal., J Immunol, 179:2509-2519, 2007). Activated T cells then localize tothe infected lung where they express various effector mechanisms. As anillustration of the importance of proper CD11c⁺ migration and function,CD11c⁺ depletion prior to M. tuberculosis infection delays the CD4⁺ Tcell response and exacerbates the outcome of infection (Tian et al., JImmunol, 175:3268-3272, 2005).

II. IL12Rβ1

IL12 family members mediate their biological activities throughspecific, high affinity dimeric receptors. These receptors all shareIL12Rβ1, a 100 kDa glycosylated protein that spans the plasma membraneand serves as a low affinity receptor for the IL12p40-subunit of IL12family members (Chua et al., J Immunology 153(1):128-36, 1994; Chua etal., J Immunology 155:4286-4299, 1995). Co-expression of IL12Rβ1 withIL12Rβ2 or IL23R results in high affinity binding of IL12 and IL23,respectively, and confers biological responsiveness to these cytokines(Parham et al., J Immunol, 168:5699-5708, 2002; Presky et al., Proc NatlAcad Sci USA, 93:14002-14007, 1996; van Rietschoten et al.,Immunogenetics, 51:30-36, 2000). Polymorphisms in IL12β or IL12Rβ1 areassociated with psoriasis (Capon et al., 2007), atopic dermatitis andother allergic phenotypes (Takahashi et al., Hum Mol Genet,14:3149-3159, 2005). Since IL12Rβ1 mediates the activity of cytokinessuch as IL-12p70, IL-23 and IL-12(p40)₂ it has the potential to impactmany aspects of the immune responses; including for example enhancingprotective immunity to pathogens as well as regulating the damagingeffects inflammatory responses associated with autoimmune pathologies(such as arthritis).

A large body of data demonstrates the essential function that theIL12Rβ1 gene serves in humans to positively regulate immunity tomycobacterial pathogens. For example, non-functional IL12Rβ1 allelespredispose an individual to mycobacterial susceptibility (Altare et al.Science, 280:1432-1435, 1998; de Jong et al., Science, 280:1435-1438,1998; Filipe-Santos et al., Semin Immunol, 18:347-361, 2006; Fortin etal., Annu Rev Genomics Hum Genet, 8:163-192, 2007). The associationbetween IL12Rβ1 deficiency and mycobacterial susceptibility undoubtedlyreflects the importance of the IL12Rβ1 gene to a wide variety of celltypes. Thus, understanding how IL12Rβ1 expression and IL12Rβ1-dependentsignaling is regulated has important implications for tuberculosis andmay impact other diseases.

In some embodiments, following M. tuberculosis infection DCs expressIL12Rβ1 and an alternatively spliced variant of IL12Rβ1 mRNA termedIL12Rβ1ΔTM mRNA. This splice variant can be detected at the mRNA levelon CD11c⁺ cells from the lungs of M. tuberculosis infected mice and as aprotein in the membrane of DCs. In contrast to IL12Rβ1, IL12Rβ1ΔTM mRNAencodes an altered C-terminal sequence and is lacking atransmembrane-domain; nevertheless the IL12Rβ1ΔTM protein is stillmembrane associated. While the expression of IL12Rβ1 mRNA is increasedduring active pulmonary tuberculosis in humans (Taha et al., Am J RespirCrit Care Med, 160:1119-1123, 1999), expression of human IL12Rβ1ΔTM mRNAduring pulmonary tuberculosis has never been assessed.

III. Alternative Splicing

Alternative splicing is emerging as an important regulator of immunity(Lynch et al., Nat Rev Immunol, 4:931-940, 2004). It is estimatedthat >75% of human genes undergo alternative splicing, many of which areexclusively expressed by the immune system (Johnson et al., Science,302:2141-2144, 2003). The list of proteins regulated by splicing includethose involved in intracellular signaling cascades (i.e. Fyn, Syk),membrane adhesion (i.e. CD31, CD44 and CD54) and cell activation (i.e.CD45 and CD152) (Lynch et al., Nat Rev Immunol, 4:931-940, 2004).Alternatively spliced cytokine receptors can regulate inflammatoryevents by functioning as either agonists or antagonists of cytokinesignaling (Levine et al., J Immunol, 173, 5343-5348, 2004). The list ofalternatively splice cytokine receptors include members of the class Icytokine receptor superfamily (IL4R, IL5R, IL6R, IL7R, IL9R, EpoR,GCSFR, GMCSFR, gp130, and LIFR), class II cytokine receptor superfamily(IFNAR1 and IFNAR2), IL-1/TLR family (IL1RII, IL1RAcP), TGF-receptorfamily (TRI, activin receptor-like kinase 7), TNFR superfamily(TNFRSF6/Fas/CD95, TNFRSF9/4-1BB/CD137 and the IL17R (Levine et al., JImmunol, 173, 5343-5348, 2004). In some embodiments, IL12Rβ1ΔTM can nowbe added to this list of spliced and functioning cytokine receptors.

The mouse IL12Rβ1 and IL12Rβ1ΔTM cDNAs were originally cloned based ontheir nucleotide homology to human IL12Rβ1 (Chua et al., J Immunology155:4286-4299, 1995). When transfected into COS cells, both cDNAsproduced proteins that bind [¹²⁵I]-IL12 with similar low affinities,suggesting that IL12Rβ1 and IL12Rβ1ΔTM proteins were both expressed onthe cell surface (Chua et al., J Immunology 155:4286-4299, 1995).However no function has been ascribed to IL12Rβ1ΔTM. In one embodiment,it is now demonstrated that while mouse IL12Rβ1ΔTM cannot substitute forIL12Rβ1, it can function in DCs to enhance IL12(p40)₂ andIL12Rβ1-dependent migration and promote CD4⁺ T cell activation inresponse to M. tuberculosis infection. In a preferred embodiment,expression of IL12Rβ1ΔTM by DCs therefore serves as a (previouslyunknown) positive-regulator of IL12Rβ1-dependent events. Selectivereconstitution of IL12Rβ1^(−/−) DCs with IL12Rβ1 and/or IL12Rβ1ΔTMdemonstrates that IL12Rβ1ΔTM can augment, but not substitute for,IL12Rβ1-dependent DC migration. In vivo relevance is demonstrated byexperiments demonstrating 1) that lung CD11c⁺ cells express IL12Rβ1ΔTMafter M. tuberculosis infection and 2) that reconstitution ofIL12Rβ1^(−/−) DCs with IL12Rβ1 and IL12Rβ1ΔTM accelerates in vivo CD4⁺ Tcell activation as compared to IL12Rβ1^(−/−) DCs reconstituted withIL12Rβ1 alone. That this may be relevant to the understanding of humanDC biology is suggested by the observation that human peripheral bloodmononuclear cell (PBMC)-derived DCs also respond to stimulation bysplicing IL12Rβ1 mRNA. Surprisingly, the stimuli that elicit IL12Rβ1mRNA splicing are broader in origin for humans than for mice.

Results demonstrate that the IL12Rβ1ΔTM protein is induced in cellsexposed to the pathogen M. tuberculosis and that it is expressed in vivoin the lungs of mice infected with this pathogen. Results furtherindicate that the IL12Rβ1ΔTM protein augments IL12 signaling by ligandsof the IL12 receptor complex and increases the chemotactic activity ofmotile cells. Since IL12 is required for induction of cellular responsesthat limit mycobacterial disease and is also required to promote type-1cellular responses, in one embodiment the IL12Rβ1ΔTM protein may be usedto augment vaccine induced IL12 expression and increase type-1 immuneresponses to vaccination.

Results also demonstrate that IL12(p40)₂ enhances M.tuberculosis-dependent NFκB activation. IL12(p40)₂ is produced bymigrating DCs (Robinson et al., J Immunol, 181:5560-5567, 2008) andIL12B sufficient DCs are more efficient at migrating to the draininglymph node and stimulating T-cell responses than IL12B deficient DCs(Khader et al., J Exp Med, 203:1805-1815, 2006; Reinhardt et al., JImmunol, 177:1618-1627, 2006; Robinson et al., J Immunol, 181:5560-5567,2008). That IL12(p40)₂ can activate NFκB-dependent events has also beenobserved in microglial cells (Dasgupta et al., Hybridoma (Larchmt),27:141-151, 2008). Since it lacks any known intracellular signalingcapacity, in one embodiment the IL12Rβ1ΔTM protein may enhanceIL12Rβ1-signaling by increasing the affinity of IL12(p40)₂ for IL12Rβ1or by forming some other structure that favors IL12(p40)₂-signaling. Inparticular, as IL12p40 binds only to dimer/oligomers of IL12Rβ1 protein(Chua et al., J Immunology 155:4286-4299, 1995), another embodiment itis possible that the IL12Rβ1ΔTM protein stabilizes oligomerization ofthe IL12Rβ1 protein.

In a preferred embodiment, these findings present a pathway whereby adeficiency in IL12Rβ1ΔTM results in an impaired ability to control M.tuberculosis infection. The expression of IL12Rβ1 mRNA and IL12Rβ1ΔTMmRNA in the CD11c⁻ fraction of M. tuberculosis-infected lungs suggeststhat the proteins encoded by these sequences may play a role during thechronic stage of infection. Results clearly indicate that for mouse DCs,both M. tuberculosis and M. avium are capable of eliciting IL12Rβ1 mRNAsplicing whereas Y. pestis stimulation is not. This may reflect theactivation of distinct Toll-like receptor (TLR) cascades. IL12Rβ1ΔTMexpression is likely not restricted to DCs and may augment functionsother than migration.

IV. Cre-Lox Recombination

Cre-Lox recombination refers to a site-specific recombinase technologyused to carry out in vivo site-specific recombination events, includingdeletions, insertions, translocations and inversions, in the genomicDNA. The cyclic recombinase (Cre) enzyme and the original Lox sitecalled the LoxP sequence are derived from a bacteriophage P1. Cre-Loxrecombination allows the DNA modification to be targeted to a specificcell type or be triggered by a specific external stimulus in both ineukaryotic and prokaryotic systems and is commonly used to circumventembryonic lethality that often occurs following the systemicinactivation of one or more genes. The recombination event is mediatedby Cre-recombinase, a site-specific enzyme that catalyzes therecombination of DNA between a pair of short target sequences called theLoxP sequences. These sequences contain specific binding sites for Crethat surround a directional core sequence where recombination can occurwithout inserting any extra supporting proteins or sequences. The resultof the recombination event depends on the orientation of the loxP sites.For two lox sites on the same chromosome arm, inverted loxP sites willcause an inversion of the intervening DNA, while a direct repeat of loxPsites will cause a deletion event. If loxP sites are on differentchromosomes it is possible for translocation events to be catalysed byCre induced recombination. Placing Lox sequences appropriately willallow genes to be activated, repressed, or exchanged for other genes.The activity of the Cre enzyme can be controlled so that it is expressedin a particular cell type or triggered by an external stimulus,including for example, a chemical signal or a heat shock. In oneembodiment, these targeted DNA changes are useful in cell lineagetracing and when mutants are lethal if expressed globally.

V. Quantitative Polymerase Chain Reaction SYBR Green Assay

SYBR green (or SYBR green I) refers to an asymmetrical cyanine dye usedas a nucleic acid stain in molecular biology. When SYBR green I binds toDNA the resulting DNA-dye-complex absorbs blue light (λ_(max)=497 nm)and emits green light (λ_(max)=520 nm). While SYBR green preferentiallybinds to double-stranded DNA, it will also bind single-stranded DNA andRNA, albeit with a lower performance than DNA. SYBR green is employed inseveral areas of biochemistry and molecular biology, including its useas a dye for the quantification of double stranded DNA in some methodsof real time PCR. It is also used to visualize DNA in gelelectrophoresis. In addition to labeling pure nucleic acids, SYBR greencan also be used for labeling of DNA within cells for flow cytometry andfluorescence microscopy. In these cases RNase treatment may be requiredto reduce background from RNA in the cells.

In molecular biology, real-time polymerase chain reaction, also calledquantitative real time polymerase chain reaction, refers to a laboratorytechnique based on standard PCR, which is used to amplify andsimultaneously quantify a targeted DNA molecule. For one or morespecific sequences in a DNA sample, real time PCR enables both detectionand quantification. The quantity can be either an absolute number ofcopies or a relative amount when normalized to DNA input or additionalnormalizing genes. The procedure follows the general principle ofpolymerase chain reaction as commonly known in the art, with the keyadditional feature being that the amplified DNA is detected as thereaction progresses (i.e. in real time). This approach differs fromstandard PCR where the product of the reaction is only detected once thereaction is completed. Two common methods for detection of products inreal-time PCR are available; the first uses non-specific fluorescentdyes (i.e. SYBR green) that bind with the double-stranded DNA generatedduring the PCR reaction, the second uses sequence-specific DNA probesconsisting of oligonucleotides that are labeled with a fluorescentreporter which permits detection only after hybridization of the probewith its complementary DNA target.

In some embodiments real-time PCR is combined with a reversetranscription reaction to quantify messenger RNA and non-coding RNA incells or tissues. In some embodiments the abbreviation “qPCR” denotesreal-time PCR while qRT-PCR denotes real-time reverse-transcription PCRis often denoted as: qRT-PCR The acronym “RT-PCR” commonly denotesreverse transcription polymerase chain reaction and not real-time PCR,but not all authors adhere to this convention

VI. Experimental

The following are examples that further illustrate embodimentscontemplated by the present invention. It is not intended that theseexamples provide any limitations on the present invention. In theexperimental disclosure that follows, the following abbreviations apply:eq. or eqs. (equivalents); M (Molar); μM (micromolar); N (Normal); mol(moles); mmol (millimoles); μmol (micromoles); nmol (nanomoles); pmoles(picomoles); g (grams); mg (milligrams); μg (micrograms); ng (nanogram);vol (volume); w/v (weight to volume); v/v (volume to volume); L(liters); ml (milliliters); μl (microliters); cm (centimeters); mm(millimeters); μm (micrometers); nm (nanometers); C (degreesCentigrade); rpm (revolutions per minute); DNA (deoxyribonucleic acid);kdal (kilodaltons).

IL12Rβ1^(−/−) is Required for M. tuberculosis-Induced DC Migration andFunction

CD11c⁺ cells are essential for the control of M. tuberculosis infection(Tian et al., J Immunol, 175:3268-3272, 2005) and IL12(p40)₂ is requiredfor their migration in response to pathogenic stimuli (Khader et al., JExp Med, 203:1805-1815, 2006; McCormick et al., J Immunol,181:2356-2367, 2008; Robinson et al., J Immunol, 181:5560-5567, 2008).Since IL12β is required for DC migration in response to M. tuberculosis(Khader et al., J Exp Med, 203:1805-1815, 2006), it was thereforenecessary to determine whether the IL12Rβ1 gene—which encodes thereceptor for IL12β (Oppmann et al., Immunity, 13:715-725, 2000; Preskyet al., J Immunol, 160:2174-2179, 1998; Wang et al., Eur J Immunol,29:2007-2013, 1999)—is expressed by DC in response to M. tuberculosisand if it is required for subsequent DC migration and T cell priming.Delivery of M. tuberculosis via the intratracheal route revealed thatthe frequency of CD11c⁺ cells expressing IL12Rβ1 in the lungs increasesthree hours after delivery (FIG. 1A). BMDCs also respond to M.tuberculosis by increasing IL12Rβ1 expression on CD11c⁺ cells (FIG. 1B).An immature population of IL12Rβ1^(−/−) BMDC was generated to determineif IL12Rβ1 was required for DC migration following mycobacterialstimulation, based on their ability to migrate towards the homeostaticchemokine CCL19 using a previously established method (Khader et al., JExp Med, 203:1805-1815, 2006). IL12Rβ1^(−/−) DCs are morphologically andphenotypically similar to C57BL/6 DCs (data not shown); however in an invitro transwell assay IL12Rβ1^(−/−) DCs had a significantly lowermigratory response towards CCL19 after exposure to varyingconcentrations M. tuberculosis compared to C57BL/6 controls (FIG. 1C).To determine if this was also true in vivo, an emulsion of M.tuberculosis and carboxyfluorescein succinimidyl ester (CFSE) wasadministered to il12b^(−/−), IL12Rβ1^(−/−), il12rb2^(−/−) and C57BL/6mice via the trachea and the number of CD11c⁺ CFSE⁺ cells in thedraining MLN was determined 18 hours later. While non-manipulated miceof all genotypes had similar numbers of CD11c⁺ cells in their lung andMLN (data not shown), a lower frequency (FIG. 1D,E) and fewer numbers(FIG. 1F) of CD11c⁺ CFSE⁺ cells in the MLN of il12b^(−/−) andIL12Rβ1^(−/−) was consistently observed in mice after administration ofM. tuberculosis and CFSE via the trachea. This was not true ofil12rb2^(−/−) mice, further supporting a role for IL12(p40)₂ and notIL12p70 in DC migration (Khader et al., J Exp Med, 203:1805-1815, 2006).These results demonstrate that IL12Rβ1 is required for M.tuberculosis-induced CD11c⁺ cell migration from the lung to the drainingMLN.

A Reduced Frequency of IL12Rβ1-Sufficient CD11c⁺ Cells in the LungDelays the Activation of M. tuberculosis-Specific T Cells

CD4⁺ T cell responses to M. tuberculosis antigens are initiated in theMLN (Gallegos et al., J Exp Med, 205:2359-2368, 2008; Reiley et al.,Proc Natl Acad Sci USA, 105:10961-10966, 2008; Winslow et al., ImmunolRev, 225, 284-299, 2008; Wolf et al., J Exp Med, 205:105-115, 2008).Therefore a delay in CD11c⁺ cell migration should delay the activationof M. tuberculosis-specific CD4⁺ T cells. To test this theory diphtheriatoxin (DT) was used to specifically deplete IL12Rβ1^(+/+) CD11c⁺ cellsfrom bone marrow chimeras that contain diphtheria toxin receptorpositive (DTR⁺) C57BL/6 CD11c⁺ cells and DTR negative IL12Rβ1^(−/−)CD11c⁺ cells. M. tuberculosis-specific T cell activation tointratracheal administration of M. tuberculosis antigen was thenmeasured. The chimeras were generated by reconstituting lethallyirradiated C57BL/6 mice with 25% IL12Rβ1^(−/−) and 75% Itgax-DTR/eGFPbone marrow (DTR:IL12Rβ1^(−/−) mice) or, as a control, 25% C57BL/6 and75% Itgax-DTR/eGFP bone marrow (DTR:WT mice). The Itgax-DTR/eGFP miceare transgenic for a simian DTR fused to an enhanced green fluorescentprotein (eGFP) that is under control of the Itgax (or CD11c) promoter.Upon DT administration, CD11c⁺ cells containing this transgene aretransiently depleted in most tissues (Jung et al., Immunity17(2):211-20, 2002).

In control DTR:WT mice injected with saline the majority of CD11c⁺ cellsare GFP⁺, demonstrating reconstitution of the lung with DTR expressingcells (FIG. 2A). Both GFP⁺ and the subset of GFP⁻ CD11c⁺ cells areIL12Rβ1^(+/+) and express basal levels of IL12Rβ1 protein on theirsurface (FIG. 2A). Upon injection of DT the frequency of GFP⁺ CD11c⁺cells drops approximately 12 fold (FIG. 2B), resulting in an increasedratio of GFP⁻ to GFP CD11c⁺ cells. Treating the DTR:IL12Rβ1^(−/−) micewith DT resulted in a similar drop in GFP⁺ CD11c⁺ cells (FIG. 2F-G) andtherefore a greatly reduced frequency of IL12Rβ1^(+/+) CD11c⁺ relativeto IL12Rβ1^(−/−) CD11c⁺ cells in the lungs of these mice.

To compare the relative T cell activating ability of lungs harboring ahigh frequency of IL12Rβ1^(+/+) CD11c⁺ cells to those with a lowfrequency, the response of antigen-specific cells in the MLN wasmeasured. To this end 1.5×10⁶ CFSE-labeled ESAT-specific CD4⁺ T cells(Reiley et al., Proc Natl Acad Sci USA, 105:10961-10966, 2008) wereintravenously transferred into DT injected DTR:WT or DTR:IL12Rβ1^(−/−)mice immediately prior to instillation via the trachea of ESAT6₁₋₂₀peptide and 1 μg of irradiated M. tuberculosis. Eighteen hours later thefrequency of ESAT-specific T cells (FIG. 2C and FIG. 2H) expressingmarkers of activation CD69 (FIG. 2D and FIG. 2I) and CD44 (FIG. 2E andFIG. 2J) in the draining MLN was determined. The frequency ofESAT6-specific T cells that expressed a high level of CD69 (FIG. 2K) andCD44 (FIG. 2L) in response to two different doses of antigen within the18 hours of the experiment was significantly lower in the mice with areduced frequency of IL12Rβ1^(+/+) CD11c⁺ cells. Thus, an increase inthe ratio of IL12Rβ1^(−/−) to IL12Rβ1^(+/+) DCs in the lungs isassociated with impaired activation of antigen-specific T cells in thedraining MLN. These data demonstrate that IL12Rβ1 expression in CD11c⁺cells within the lung is required for M. tuberculosis-induced DCmigration and induction of T cell activation in vivo.

IL12(p40)₂ Initiates Nuclear Accumulation of NF-κB in DCs

There is a need to better understand the mechanism by whichIL12Rβ1-dependent signaling modulates DC chemotaxis following exposureto M. tuberculosis. Lower levels of CCR7 (the receptor for CCL19) do notaccount for this result, as surface expression of CCR7 is similarbetween activated wild type and IL12Rβ1^(−/−) BMDCs (data not shown). Todetermine if any intracellular signaling pathways that influence DCmigration were altered in IL12Rβ1^(−/−) DCs, phosphorylation levels ofNF-κB, SAPK/JNK, p38α MAP Kinase and STAT3 were measured in these cellsfollowing stimulation with M. tuberculosis. Results demonstrated thatstimulation of C57BL/6 DCs increases phospho-NF-κB levels above those ofunstimulated controls (FIG. 3A, B). However, levels of phospho-NF-κBwere consistently observed to be lower in IL12Rβ1^(−/−) DCs compared towild type DCs at several time points despite equivalent levels of totalNF-κB (FIG. 3C, D). No differences in phospho-SAPK/JNK, p38α MAP Kinaseand STAT3 were observed between wild type and IL12Rβ1^(−/−) DCs (datanot shown). These data suggest that NFκB dependent processes arecompromised in IL12Rβ1^(−/−) DCs.

Since NFκB phosphorylation was defective in IL12Rβ1^(−/−) DCs, it wasreasoned that NFκB binding should be enhanced when DCs are stimulatedvia IL12Rβ1. To test this hypothesis il12b^(−/−) BMDC were exposed to M.tuberculosis and/or IL12(p40)₂ for 1 hour and the amount of NFκBconsensus sequence-binding proteins in nuclear extracts of the treatedcells was compared via electromobility shift assay (EMSA). il12b^(−/−)BMDCs were used for this experiment to eliminate potential backgroundNFκB activation from endogenous IL12(p40)₂. FIG. 3E demonstrates thatthe addition of M. tuberculosis to DC cultures increases the nuclearaccumulation of NFκB over that seen in untreated BMDC (compare lanes 1and 3). IL12(p40)₂ was also sufficient to increase the nuclearaccumulation of NFκB over that seen in untreated BMDC (compare lanes 1and 7). The addition of both M. tuberculosis and IL12(p40)₂synergistically augmented NFκB activation above that of each stimulusalone (compare lanes 3 and 7 to lane 5). Thus, the data demonstrate thatIL12(p40)₂ is able to stimulate NFκB nuclear migration in DCs.Consequently, the failure of IL12Rβ1^(−/−) DCs to migrate (FIG. 1-2)associates with impaired NFκB-dependent gene activation.

BMDCs Express IL12Rβ1 mRNA and an IL12Rβ1 mRNA Alternative SpliceVariant after Exposure to M. tuberculosis

While DC's are not a commonly acknowledged target for the inflammatorycytokine IL-12, reports have indicated that the subunits of the receptorfor IL-12 are expressed in these cells. The expression of IL12Rβ1ΔTMmRNA by DCs has not been universally accepted due to an inability toreproducibly detect this transcript (Grohmann et al., Immunity9:315-323, 1998); including the inability to amplify the IL12Rβ1transcript with primers spanning the distal portion of exon 16. However,given the influence of IL12Rβ1 on DC migration (FIG. 1-2) IL12Rβ1 mRNAexpression in these cells was re-examined. Results indicate that carefuluse of primer sets allows for detection of an IL12Rβ1 gene product inthese cells. The murine IL12Rβ1 gene is located on autosomal chromosome8C2 at position 73.737.483-73.750.411 and comprises 16 exons (FIG. 4A;NCBI GeneID 16161). Upon transcription and intron-removal, exons 1-13are translated into the extracellular portion of the IL12Rβ1 protein,while exon 14 and exons 15-16 are translated into the transmembrane (TM)and intracellular portions, respectively (FIG. 4B). To determine thetranscription activity of this gene in DCs, cDNA from BMDC cultures wereamplified with a variety of primers spanning different lengths ofIL12Rβ1 cDNA (FIG. 4B; forward and reverse primers are indicated by ∇-and Δ-arrows, respectively). Amplification with primers (P) recognizingthe extracellular-encoding region (P1-P2) resulted in an amplicon (FIG.4C); cDNA from concanavalin-A activated splenocytes is used as apositive control (IL12Rβ1^(+/+)). Confirming the results of Grohmann etal. (Immunity 9:315-323, 1998), amplification of DC cDNA with primersthat recognize the intracellular encoding region (P3-P6) did not resultin a visible amplicon from DC cDNA. However, amplification of a more 3′region with primers P3-P5 did result in a PCR product in bothunstimulated and stimulated DCs. Surprisingly, under these amplificationconditions a second smaller band was also observable, but only in DCsthat had been stimulated with M. tuberculosis (see arrow, FIG. 4C). Thissecond band does not appear upon amplification with primers that spanthe TM-encoding region (P3-P4). Sequencing both the larger and smallerband amplified by primers P3-P5 revealed that the larger productrepresents IL12Rβ1 mRNA and that the smaller product is identical exceptfor a 97-bp deletion (FIG. 4D). This deletion has two effects: (1)Deletion of the TM sequence encoded by exon 14 and (2) a translationalframe shift that results in an early stop codon. This translationalframe shift also results in the loss of the Box1/2 signaling domainsthat are found in the IL12Rβ1 protein (van de Vosse et al.,Immunogenetics 54:817, 2003). Both the nucleotide and deduced amino acidsequence of this smaller band (FIG. 4E) match that of a previouslyreported alternative splice variant of the mouse IL12Rβ1 transcript(Chua et al., J Immunology 155:4286-4299, 1995). Thus, DCs respond to M.tuberculosis by expressing two species of IL12Rβ1 mRNA: atransmembrane-containing transcript referred to as IL12Rβ1 mRNA and analternatively spliced variant of IL12Rβ1 mRNA referred to as IL12Rβ1ΔTMmRNA. It is interesting that IL12Rβ1ΔTM remains membrane-associateddespite the absence of a transmembrane-domain. It is believed thatIL12Rβ1ΔTM also functions to enhance IL12Rβ1-dependent processes in Tand NK cells.

Kinetics of BMDC IL12Rβ1ΔTM mRNA Expression Following Exposure to M.tuberculosis

Attempts to quantify IL12Rβ1ΔTM mRNA expression induced in BMDCs by M.tuberculosis proved difficult due to an inability to design a Taqmanreal time PCR probe that recognized IL12Rβ1ΔTM cDNA and not IL12Rβ1cDNA. To better quantify the kinetics of IL12Rβ1ΔTM mRNA expressionrelative to IL12Rβ1 mRNA in M. tuberculosis-stimulated DCs, a PCR-basedassay was developed—hereafter referred to as “IL12Rβ1 Spectratypeanalysis”. IL12Rβ1 Spectratype analysis is akin to TCR-CDR3 Spectratypeanalysis (Pannetier et al., Proc Natl Acad Sci USA, 90:4319-4323, 1993)and is described in FIG. 5. When IL12Rβ1 Spectratype analysis wasapplied to BMDCs, a dose-dependent increase in the ratio of IL12Rβ1ΔTMmRNA to IL12Rβ1 mRNA was observed after a 3-hour exposure to M.tuberculosis (FIG. 6A). In contrast IL12Rβ1 mRNA remains the dominanttranscript in unstimulated DCs for up to 6-hours (FIG. 6A). Western blotanalysis demonstrated that IL12Rβ1 is the dominant protein product inunstimulated cells while IL12Rβ1ΔTM protein increases in abundancefollowing M. tuberculosis stimulation (FIG. 6B). That IL12Rβ1ΔTM proteincould locate in the membrane was indicated by Western blot analysis ofcellular fractions (FIG. 5H). Thus, analysis of mRNA and Western blotanalysis confirm that DCs increase the expression of IL12Rβ1ΔTM mRNA andproduction of IL12Rβ1ΔTM protein following exposure to M. tuberculosis.

To assess the specificity of IL12Rβ1 mRNA splicing in response to M.tuberculosis, DCs were stimulated with a variety of other microbial andcytokine stimuli. Specifically, DCs were stimulated with M. avium and Y.pestis (at an identical MOI) as well as with M. avium cell wall extract,Escherichia coli lipopolysaccharide (LPS), TNFα, IL12 and IL12(p40)₂.Production of IL12Rβ1ΔTM mRNA was subsequently assessed by IL12Rβ1Spectratype analysis. Both M. avium and Y. pestis were capable ofactivating DCs as measured by IL12p40 production (FIG. 7A). As shown inFIG. 7B, over a 3-hour incubation M. avium was capable of elicitingIL12Rβ1ΔTM production with kinetics that were similar to that elicitedby M. tuberculosis. This was also observed with M. avium cell wallextract (FIG. 7C). Stimulation with Y. pestis and purified LPS (FIG.7D-E) failed to generate IL12Rβ1ΔTM over the same 3-hour period.Negative results were also obtained with TNFα, IL12 andIL12(p40)₂-stimulated DCs (FIG. 7F-H). Thus, DCs increase the expressionof IL12Rβ1ΔTM not only in response to M. tuberculosis but also to therelated organism M. avium; stimulation with gram negative Y. pestis,purified LPS and cytokines TNFα, IL12 and IL12(p40)₂ fails to elicitthis same response.

Human DCs Respond to Stimuli by Splicing IL12Rβ1

Following their activation, human DCs increase surface expression ofIL12Rβ1 (Nagayama et al., J Immunol, 165:59-66, 2000). Two isoforms ofthe human IL12Rβ1 mRNA transcript are reported in publicly availabledatabases: full length IL12Rβ1 (isoform 1; Swiss-Prot ID P42701-1) and ashorter isoform that is the product of alternative splicing (isoform 2;Swiss-Prot P42701-3). These sequences are available athttp://www.uniprot.org/uniprot/P42701 and are reproduced in FIG. 8A-B.To determine if human DCs splice the IL12Rβ1 transcript followingstimulation in a manner that is analogous to mouse DCs, monocyte-derivedDCs were exposed to a variety of stimuli, some of which are knowninducers of DC IL12Rβ1 expression (Nagayama et al., J Immunol,165:59-66, 2000). cDNAs generated from the stimulated DCs were assessedfor the relative levels of transcripts for IL12Rβ1 isoforms 1 and 2using specific primers; cDNA from CD3⁺ PBMCs was used as a positivecontrol. All samples (including DCs exposed to media alone) expressedIL12Rβ1 when assayed with primers that recognized both isoforms 1 and 2(FIG. 8C, top panel). However, amplification with primers specific toeither isoform 1 (FIG. 8C, middle panel) or isoform 2 (FIG. 8C, bottompanel) revealed that expression of these two transcripts wasdifferentially regulated depending on the stimulus. Specifically, theproduction of isoform 2 was strongly associated with exposure to LPS,IL1β, IL2 and CCL3. Stimulation of human DCs with M. tuberculosis alsoelicited expression of IL12Rβ1 isoform 2 over a 6-hour time course (FIG.8D). These experiments demonstrate that human DCs, like mouse DCs,respond to specific stimuli by splicing the IL12Rβ1 transcript.

IL12Rβ1 mRNA and IL12Rβ1ΔTM mRNA are Expressed by CD11c⁺ Cells in the M.tuberculosis-Infected Lung

IL12Rβ1ΔTM mRNA expression in response to M. tuberculosis infection invivo was also examined by comparing the relative abundance of IL12Rβ1ΔTMmRNA to IL12Rβ1 mRNA over a time course in the lungs of miceaerogenically infected with M. tuberculosis. IL12Rβ1ΔTM abundance wasanalyzed in aerosol M. tuberculosis infected mice using a modified TCRCDR3 spectratyping assay with the ability to quantitate the relativeratios of two or more transcript sizes. In uninfected controls, theexpression of IL12Rβ1ΔTM mRNA was minimal over the entire 30-day period,with IL12Rβ1 mRNA being the dominant transcript observed (FIG. 9A). InM. tuberculosis-infected animals, however, a shift in the ratio ofIL12Rβ1ΔTM mRNA to IL12Rβ1 mRNA in the lung is observed at 9 dayspost-infection (FIG. 9B), with IL12Rβ1ΔTM mRNA reaching 2.4-fold higherabundance than IL12Rβ1 mRNA in some cases. After days 9-12, the ratio ofIL12Rβ1ΔTM mRNA to IL12Rβ1 mRNA in the lung diminished but stillremained higher than that of uninfected controls up to day 30. Thisresult was observed in several independent experiments (FIG. 9C). In theliver (an organ distal to the initial site of infection) elevatedbaseline levels of IL12Rβ1ΔTM mRNA expression compared to the lung wereobserved (FIG. 9D); however these levels remained unchanged through theearly course of M. tuberculosis infection (FIG. 9D). In summary,IL12Rβ1ΔTM mRNA is expressed subsequent to M. tuberculosis infection invivo—the relative ratio to IL12Rβ1 mRNA being dependent upon timepost-infection.

The expression of IL12Rβ1ΔTM by DCs in vitro (FIG. 6) and by the M.tuberculosis-infected lung in vivo (FIG. 9) prompted experiments todetermine whether CD11c⁺ cells from M. tuberculosis-infected lungs arethe source of this transcript. CD11c⁺ cells from the lungs of M.tuberculosis-infected mice were isolated by magnetic beads at varioustime points after infection and expression of IL12Rβ1ΔTM mRNA wasdetermined as described in FIG. 9. CD11c⁺ cells from M.tuberculosis-infected mice consistently expressed a higher ratio ofIL12Rβ1ΔTM mRNA to IL12Rβ1 mRNA compared to those isolated fromuninfected controls, the highest being observed at days 7-12post-infection (compare top panels of FIG. 10A-B). Notably, the CD11c⁻cells from M. tuberculosis-infected mice also expressed a higher ratioof IL12Rβ1ΔTM mRNA to IL12Rβ1 mRNA compared to uninfected controls, thehighest being observed at days 20 and 30 post-infection (compare lowerpanels of FIG. 10A-B). This result was observed in several independentexperiments (FIG. 10C-D). These data demonstrate that following low doseaerogenic M. tuberculosis infection, lung CD11c⁺ cells exhibit increasedexpression of IL12Rβ1ΔTM mRNA and that CD11c⁻ cells can also expressthis transcript as infection progresses.

Similar to M. tuberculosis, M. avium and Y. pestis are lung-tropicintracellular pathogens. Since exposure to M. avium, but not Y. pestis,increased DC expression of IL12Rβ1ΔTM mRNA, it was next determinedwhether IL12Rβ1ΔTM mRNA was also expressed in the M. avium or Y. pestisinfected lung. Mice were aerogenically infected with M. avium (FIG. 11A)or intranasally with Y. pestis KIMD27 (FIG. 11C) and a time course ofIL12Rβ1ΔTM mRNA abundance relative to IL12Rβ1 mRNA was performed. Aswith M. tuberculosis, a shift in the ratio of IL12Rβ1ΔTM mRNA to IL12Rβ1mRNA was observed in the lung at 9 days post-infection (FIG. 11B).Despite similar numbers of CFU at day 4 post-infection, only one out offive Y. pestis infected animals showed expression of IL12Rβ1ΔTM mRNA(FIG. 11D, top panels). Negative results were also obtained uponinfection with the more immunostimulatory strain Y. pestis KIMD27/pLpxL(FIG. 11D, bottom panels). Thus, in addition to M. tuberculosis (FIG.11) expression of IL12Rβ1ΔTM mRNA in the lungs is also elicited by M.avium—but not Y. pestis.

IL12Rβ1ΔTM Enhances IL12Rβ1-Dependent Migration

DCs exhibit IL12(p40)₂ and IL12Rβ1-dependent migration in response to M.tuberculosis after only a 3 hour exposure to this organism (FIG. 1C andKhader et al., J Exp Med, 203:1805-1815, 2006; Robinson et al., JImmunol, 181:5560-5567, 2008). Given that IL12Rβ1ΔTM mRNA is transcribedand translated within this timeframe (FIG. 6), and considered along withthe ability of the IL12Rβ1ΔTM protein to bind the related protein IL12,the contribution of IL12Rβ1ΔTM to M. tuberculosis-induced,IL12(p40)₂-dependent DC migration was examined.

To address this issue an IL12(p40)₂-dependent NIH/3T3 migration thatmodels IL12(p40)₂-dependent DC migration using the commerciallyavailable NIH/3T3 mouse embryonic fibroblast cell line was used.Specifically, Russell et al. observed that NIH/3T3 cells transfectedwith IL12Rβ1 migrate towards IL12(p40)₂ while those that lack IL12Rβ1 donot. NIH/3T3 cells were split into four groups, and were transfectedwith either IL12Rβ1 alone, IL12Rβ1ΔTM alone, both IL12Rβ1 andIL12Rβ1ΔTM, or an empty vector control. All groups were cotransfectedwith eGFP to positively identify transfectants. Twenty-four hours laterall groups were placed in the upper well of a Boyden chamber; the bottomwell contained either IL12(p40)₂ or media alone. Enumerating the GFP⁺cells that migrated across the transwell allows IL12Rβ1ΔTM influencedtransfectant migration toward IL12(p40)₂ to be determined. Resultsdemonstrate that NIH/3T3 migration using this assay was both IL12(p40)₂and IL12Rβ1-dependent (FIG. 13 a). When IL12Rβ1ΔTM is substituted forIL12Rβ1, NIH/3T3 migration returns to media-alone levels. Howeverco-transfection of IL12Rβ1ΔTM alongside IL12Rβ1 resulted in anapproximate 50% increase in transfectant migration. These results arestatistically significant and have been observed across severalexperiments (FIG. 13). Thus, while IL12Rβ1ΔTM cannot substitute forIL12Rβ1 it can augment IL12Rβ1-dependent NIH/3T3 cell migration.

Related experiments were performed by selectively restoring mRNAs thatencode IL12Rβ1, IL12Rβ1ΔTM, or both IL12Rβ1 and IL12Rβ1ΔTM toIL12Rβ1^(−/−) DCs which contain a genomic insertion of the neomycinresistance gene (neo insertion) that disrupts exons 1-3 of the IL12Rβ1locus (Wu et al., J Immunol, 159:1658-1665, 1997) and thus lacks boththese proteins (FIG. 4). mRNA encoding GFP co-transfected with thespecific mRNAs via electroporation demonstrated that an antibodyspecific for the common extracellular portion of IL12Rβ1 and IL12Rβ1ΔTMonly labeled GFP⁺ CD11c⁺ cells if mRNAs for either IL12Rβ1 or IL12Rβ1ΔTMwere delivered to the IL12Rβ1^(−/−) DCs (FIG. 12A and FIG. 12B).Following stimulation with M. tuberculosis, IL12Rβ1^(−/−) DCstransfected with GFP and IL12Rβ1 were capable of migrating toward CCL19whereas those transfected with GFP alone were not (FIG. 12C).IL12Rβ1^(−/−) DCs transfected with GFP and IL12Rβ1ΔTM had migratorylevels equivalent to those transfected with GFP alone. However,co-transfection with GFP, IL12Rβ1 and IL12Rβ1ΔTM resulted in a greaterchemotaxis index than when DCs were transfected with GFP and IL12Rβ1.The majority of migrated cells were GFP positive suggesting thatmigration required transfection of the migrating cell and was not anindirect effect (FIG. 12D). These data demonstrate that IL12Rβ1ΔTM canenhance IL12Rβ1-dependent DC migration.

Finally, given that IL12Rβ1ΔTM enhanced IL12Rβ1-dependent DC migrationin vitro, it was determined whether its expression in DCs acceleratedthe activation of M. tuberculosis-specific T cells in vivo.IL12Rβ1^(−/−) BMDCs were selectively restored with mRNAs for IL12Rβ1,IL12Rβ1ΔTM, or both IL12Rβ1 and IL12Rβ1ΔTM as described above. Followingtheir electroporation and overnight culture, DCs were cultured withirradiated M. tuberculosis and ESAT₁₋₂₀ peptide for 3 hrs. After thisperiod DCs were washed and instilled via the trachea into the lungs ofC57BL/6 mice that had previously received 5×10⁶ CFSE-labeled ESAT-TCRCD4⁺ cells. Twelve hours after DC instillation the surface expression ofCD44 and CD69 by CFSE⁺ CD4⁺ cells in the draining MLN was assessed byflow cytometry. As anticipated, mice that received sham electroporatedIL12Rβ1^(−/−) DCs had fewer activated M. tuberculosis-specific T cellsin the draining MLN relative to those that received sham electroporatedC57BL/6 DCs (FIG. 12E). Restoration of IL12Rβ1 alone to IL12Rβ1^(−/−)DCs elevated the frequency of CD44^(hi) and CD62L^(lo) M.tuberculosis-specific T cells, however restoring IL12Rβ1ΔTM alone toIL12Rβ1^(−/−) DCs did not elevate the frequency of activated M.tuberculosis-specific T cells. Importantly, only when both IL12Rβ1 andIL12Rβ1ΔTM were restored to IL12Rβ1^(−/−) DCs did the frequency ofactivated M. tuberculosis-specific T cells return the level seen in micethat received C57BL/6 DCs. This result was observed across severalindependent experiments (FIG. 12F and FIG. 12G). These data demonstratethat IL12Rβ1ΔTM acts as a positive-regulator to enhanceIL12Rβ1-dependent DC migration from the lung and IL12Rβ1-dependentactivation of M. tuberculosis-specific T cells in the lung draining MLN.

IL12Rβ1ΔTM Enhances Other IL12Rβ1-Dependent Events

To determine whether IL12Rβ1ΔTM enhances other IL12Rβ1-dependent eventsan adapted STAT4-reporter assay was used. Following phosphorylation byIL12Rβ2 in an IL12-dependent manner, STAT4 translocates to the nucleuswhere it functions as a transcription factor for genes containing agamma-activated sequence (i.e. GAS) promoter. In this assay NIH/3T3cells are transiently transfected with plasmids that constitutivelyexpress IL12Rβ1, IL12Rβ2 and STAT4. STAT 4 phosphorylation is measuredafter addition of IL12 using a STAT4-reporter plasmid that containsfirefly-luciferase under the GAS-promoter. Constitutively expressedRenilla luciferase is used to normalize for transfection efficiency.STAT4 activity was observed to be both IL12- and IL12Rβ1-dependent (FIG.13 b). When IL12Rβ1ΔTM is substituted for IL12Rβ1, STAT4 activationreturns to media-alone levels. However co-transfection of IL12Rβ1ΔTMalongside IL12Rβ1 and IL12Rβ2 results in an approximate 30% increase inSTAT4 activity. These results are statistically significant and havebeen observed across three independent experiments. Collectively theseexperiments suggest that IL12Rβ1ΔTM functions in transfected NIH/3T3cells to enhance IL12Rβ1-dependent signaling.

Mice Unable to Generate the IL12Rβ1 Isoform 2 Allow Increased BacterialGrowth in the Spleen.

Data obtained using a genetically engineered mouse that can expressIL12Rβ1 isoform 1 but not IL12Rβ1 isoform 2 demonstrate that IL12Rβ1isoform 2 aids in control of the bacterial burden in the spleen (FIG.14). Since the mouse IL12Rβ1 gene is located on autosomal chromosome 8two copies of the gene are present. There are 16 exons in the codingsequence of IL12Rβ1 isoform 1, with exons 1-13 encoding theextracellular portion of the protein, exon 14 encoding the transmembraneportion and exons 15 and 16 encoding the intracellular portion of theprotein. The IL12Rβ1 isoform 2 contains a deletion of the last base ofexon 13, the complete deletion of exon 14 followed by a frame shift andgeneration of a premature stop codon. The protein produced by IL12Rβ1isoform 2 therefore lacks the transmembrane and intracellular portionsof the full-length protein. While an understanding of the mechanism ofthe invention is not necessary, and without limiting the invention toany particular mechanism, as intron sequences are thought to directsplicing one embodiment of the present invention contemplates thatdisruption of the intron sequences between exon 13 and 16 disruptsgeneration of IL12Rβ1 isoform 2. The inclusion of a LoxP site in theknock-in gene allows for targeted or conditional homologousrecombination of the inserted allele and the loss of the ability togenerate IL12Rβ1 isoform 2. CD4-cre and CD11c-cre were bred to theIL12Rβ1 isoform 2 knock-in mice. When CD4⁺ cells from the resultingCD4crexIL12Rβ1-isoform 2 mice were stimulated with concanavalin A theyfailed to generate the splice variant as determined by PCR (copy numberis reduced by 200 fold). When the CD11c and the CD4 cre mice werecrossed to the IL12Rβ1 isoform 2 knock-in mice and the resulting progenyinfected with M. tuberculosis, they exhibited increased bacterial growthin the spleen relative to IL12Rβ1 isoform 2 knock-in mice that were notcrossed to any cre expressing mice (FIG. 14). This data demonstratesthat even at low challenge doses the IL12Rβ1 isoform 2 has a role incontrolling bacterial burden in the peripheral organs of infected mice.

Human Peripheral Blood Cells from Patients with Active TuberculosisTranscribe More IL12RB Isoform 2 than Peripheral Blood Cells fromPatients with Latent Tuberculosis and Patients Undergoing SuccessfulTreatment.

A decrease in the level of the alternative splice variant indicates thatthere are fewer active bacteria driving infected cells to produce thismolecule. Thus, M. tuberculosis actively drives and manipulates theimmune response in order to achieve a successful infection. The humanmicroarray for transcriptional analysis has three probes for the IL12Rβ1gene two of which target IL12Rβ1 isoform 1 and one of which(IL12Rβ1/NM_(—)153701) targets IL12Rβ1 isoform 2. RNA from humanperipheral blood cells was analyzed using the Affymetrix humantranscriptional array and the signal for IL12Rβ1/NM_(—)153701 wascompared for the different groups of samples. Data obtained followingmicroarray transcriptional analysis of peripheral blood demonstrate thatsamples from individuals with active tuberculosis have more of IL12Rβ1isoform 2 than samples from people with latent tuberculosis or those whoare being successfully treated for disease. As depicted in FIG. 15,increased detection by this probe is evident in patients with activetuberculosis (FIG. 15, left panel), while the loss of signal duringtreatment is evident (FIG. 15, right panel).

Human Dendritic Cells Express the IL12Rβ Isoform 2 Upon Exposure to LiveM. tuberculosis.

Data obtained from an assay that quantifies the amount of IL12Rβ1isoform 1 and IL12Rβ1 isoform 2 mRNA demonstrate that the ratio oftranscription of IL12Rβ1 isoform 1 to IL12Rβ1 isoform 2 by HPBDCs isinverted within the first few hours of exposure to live M. tuberculosis(FIG. 16). Data presented herein indicate that a rapid increase intranscription of IL12Rβ1 isoform 2 occurs in human dendritic cells inresponse to live bacteria (FIG. 16) and humans with active diseaseexpress this same isoform strongly (FIG. 15). Together these datasuggest that in humans, live and growing M. tuberculosis driveexpression of IL12Rβ1 isoform 2. These results further indicate that theability to differentially detect the relative levels of two very similarisoforms of via PCR is crucial to since the level of IL12Rβ1 isoform 2relative to IL12Rβ1 isoform 1 is indicative of disease state andeffectiveness of treatment.

The assay is a quantitative polymerase chain reaction SYBR green assayin which the mRNA copy number of both IL12Rβ1 isoform 1 and 2 isdetermined as follows: A 25 μl SYBR green reaction is used with cDNAderived from monocyte derived dendritic cells, and the following: 0.4 mMprimers: 12-F and 12/13-R for amplification of IL12Rβ1 isoform 1, 9/9b-Fand 9b-R for amplification of IL12Rβ1 isoform 2 (sequences listed inTable I). The parameters for these reactions are: incubation at 95° C.for 3 min (1 cycle); denaturation at 95° C. for 10 s, annealing at 60°C. for 30 s, and extension at 72° C. for 30 s (40 cycles). IL12Rβ1isoform 1 and IL12Rβ1 isoform 2 cDNA was cloned into the plasmidpcDNA3.1 and linearized to serve as standards to calculate mRNA copynumber from the threshold cycle (Ct) values obtained from the SYBR greenassay. Melting curve analysis was used to determine amplificationspecificity and was performed immediately following the amplificationreaction. The Ct value was first normalized against the humanglyceraldehyde 3-phosphate dehydrogenase gene (GAPDH) and the relativeabundance of IL12Rβ1 isoform 1 to IL12Rβ1 isoform 2 was determined bydividing the number of IL12Rβ1 isoform 1 mRNA copies by the number ofIL12Rβ1 isoform 2 mRNA copies in a single cDNA sample.

VII. Materials and Methods

Mice

Mice were bred at the Trudeau Institute and were treated according toNational Institutes of Health and Trudeau Institute Animal Care and UseCommittee guidelines. C57BL/6, B6.129S1-Il12b^(tm1jm)/J (i.e.il12b^(−/−) mice (Magram et al., Immunity, 4:471-481, 1996),B6.129S1-Il12b^(tm1jm)/J (i.e. il12rb2^(−/−) mice (Wu et al., J Immunol,165:6221-622, 2000), and B6.FVB-Tg (Itgax-DTR/eGFP)57Lan/J (i.e.CD11c-DTR) (Jung et al., Immunity 17(2):211-20, 2002) mice wereoriginally purchased from Jackson Laboratory (Bar Harbor, Me.). C57BL/6mice deficient of the B6.129S1-Il12b^(tm1jm)/J (IL12Rβ1^(−/−) mice) havebeen described (Wu et al., J Immunol, 159:1658-1665, 1997) as haveESAT6₁₋₂₀ specific T cell receptor (TCR)-transgenic mice (Reiley et al.,Proc Natl Acad Sci USA, 105:10961-10966, 2008). Genetically modifiedmice capable of expressing IL12Rβ1 isoform 1 but not IL12Rβ1 isoform 2were provided by Taconic Artemis GmbH (Köln, Germany). Briefly, anIL12Rβ1 conditional Knock-In/Knock-Out mouse model was generated byintroducing a cDNA molecule encoding a portion of the endogenous IL12Rβ1gene downstream of the last protein-coding exon to generate an IL12Rβ1Conditional Knock-In/Knock-Out allele. The steps performed by TaconicArtemis included a) locus characterization, b) conditionalKnock-In/Knock-Out targeting vector construction and DNA sequencing, c)transfection of C57BL/6ES cells with validated targeting vector, d)isolation of targeted ES cell clones and molecular validation bySouthern analysis with external and internal probes, e) injection oftargeted ES cells into diploid blastocysts, f) chimera generation and g)crossbreeding of chimeric mice with flp deleter for in vivo selectionmarker deletion in order to generate mice heterozygous for theconditional Knock-In/Knock-Out mouse. Following generation of the mousemodel at least two F1 breeding pairs of C57BL/6 mice heterozygous forthe desired mutation were transferred to the Trudeau Institute.

Cell Preparations

M. tuberculosis infections were performed and the lung tissue and lymphnodes were processed as described previously (Khader et al., NatImmunol, 8:369-377, 2007). Single cell suspensions were prepared fromeither digested lung tissue or lymph nodes by direct dispersal through a70-μm nylon tissue strainer (BD Falcon). The resultant suspension wastreated with Geys solution (155 mM NH₄Cl, 10 mM KHCO₃) to remove anyresidual red blood cells, washed twice with complete media, counted andstained for subsequent flow cytometric analysis.

Bone Marrow-Derived Dendritic Cells

BMDCs were generated from bone marrow of 4-5 week old C57BL/6 miceharvested via perfusion of the femur and tibia medullary cavities withice cold DMEM. Marrow suspensions were pelleted and incubated in Geyssolution to lyse red blood cells. The marrow was then resuspended at4×10⁵ cells/mL in complete supplemented DMEM (cDMEM). 5 mL of bonemarrow homogenate was plated in a Petri dish (Corning Inc., Corning,N.Y.) along with 5 mL of 40 ng/mL recombinant murine GM-CSF (Peprotech,Rocky Hill, N.J.) in cDMEM solution for a final concentration of 20ng/mL GM-CSF. Cultures were maintained at 37° C. and 10% CO₂ for 3 days,at which time an additional 10 mL of 20 ng/mL GM-CSF in cDMEM was added.At 6 days, non-adherent cells were collected and the presence of CD11c⁺cells confirmed by flow cytometric analysis. For indicated experimentsCD11c⁺ cells were positively selected by magnetic purification. In thesecases 1×10⁶ CD11c⁺ cells were placed in a 2 mL culture with or withoutindicated concentrations of irradiated M. tuberculosis, Y. pestis, M.avium, TNFα, IL12 or IL12(p40)₂ in cDMEM for varying amounts of time at37° C. and 10% CO₂. After this period, cells were collected and eitherlysed for RNA and/or protein as indicated or used for chemotaxismeasurements.

Flow Cytometry

All antibodies used for flow cytometric analysis were purchased from BDPharmingen (San Diego, Calif., USA) or eBiosciences (San Diego, Calif.,USA). Experimental cells were washed with FACS buffer (2% FCS in PBS),F_(c) receptors were blocked using anti-CD16/CD32 (BD Pharmingen. Clone2.4G2) for 15 minutes and cells were stained with antibodies thatrecognize CD11c (clone HL3), I-A^(b) (clone AF6-120.1) and IL12Rβ1(CD212, clone 114). For all surface markers, positive staining wasestablished using appropriate isotype controls. Data were acquired usinga FACSCalibur (BD Biosciences, San Jose, Calif.) and analyzed withFlowJo software (Tree Star Inc., Ashland, Oreg.).

In Vitro Chemotaxis Measurement

BMDCs were activated with indicated concentrations of irradiated M.tuberculosis and their ability to respond to the chemokine CCL19 (25ng/mL; R&D Systems) was determined using the previously described invitro transwell chemotaxis assay (Khader et al., J Exp Med,203:1805-1815, 2006).

In Vivo Tracking of Lung CD11c⁺ DCs

C57BL/6, il12b^(−/−), IL12Rβ1^(−/−) and il12rb2^(−/−) mice received asuspension of 5 μg of irradiated M. tuberculosis in a 5-mM CFSE(Invitrogen) solution delivered via the trachea. Eighteen hours afterinstillation, the draining MLN were harvested, and single cellsuspensions were prepared. Flow cytometry was used to determine thefrequency and total number of CFSE-labeled CD11c⁺ cells that hadaccumulated within the MLN.

Bone-Marrow Chimeras

To generate mice in which only CD11c⁺ cells were deficient of IL12Rβ1,mixed bone marrow chimeras were generated comprising irradiated C57BL/6hosts reconstituted with 75% CD11c-DTR/25% IL12Rβ1^(−/−) bone marrow.Intraperitoneal (i.p.) injection of DT resuspended in sterile PBStheoretically removes CD11c⁺ cells expressing the DTR leaving (in thiscase) only IL12Rβ1^(−/−) CD11c⁺ cells. Briefly, 6-10 week old C57BL/6hosts were lethally irradiated with 950 Rads (i.e. a split dose of 475Rads each, four hours apart). The irradiated hosts then received 1×10⁷whole bone marrow donor cells comprising either 75% CD11c-DTR/25%IL12Rβ1^(−/−) bone marrow or 75% CD11c-DTR/25% C57BL/6 bone marrow as acontrol. Bone marrow was prepared as described above. Mice were allowedat least 6 weeks to reconstitute. Prior to ESAT₁₋₂₀ /M. tuberculosisinstillation, all mice received an i.p. injection of 4 ng DT/g of bodymass to ablate DTR-transgenic CD11c⁺ cells.

Cell Culture

For the generation of concanavalin-A blasts, C57BL/6 spleens weredispersed through a 70 μm nylon cell strainer (BD Biosciences, BedfordMass.) and the cellular homogenate pelleted (270 g, 6 min at 4° C.) andresuspended in 2 mL of Geys solution to remove red blood cells.Splenocytes were washed and resuspended at 20×10⁶ cells/mL in cDMEM and1 mL of splenocytes was plated in 6-well dishes (Corning Inc., Corning,N.Y.) along with 1 mL of 10 μg/mL concanavalin-A in cDMEM solution(Sigma-Aldrich, St. Louis, Mo.) for a final concentration of 5 ng/mLconcanavalin-A. Cultures were maintained at 37° C. and 10% CO₂ for 3days before cells were harvested for RNA and/or protein as indicated.

RNA Purification and cDNA Synthesis

Total RNA was isolated from indicated tissues and/or cell populationsusing the RNeasy method (Qiagen) and was treated with DNAse (Ambion).cDNA was subsequently synthesized using SuperScript II reversetranscription PCR kit (Invitrogen) with random hexamer primers.

PCR

To amplify the IL12Rβ1 transcript primer pairs were used thatselectively amplify the extracellular, transmembrane or intracellularencoding-portions. The relative positions of these primers (labeledP1-P6) are illustrated in FIG. 4B. P1-4 sequences are taken directlyfrom a previous report of IL12Rβ1 expression in DCs (Grohmann et al.,Immunity 9:315-323, 1998). The 5′-3′ sequences of these and the otherprimers used in this study are as follows: P1 [SEQ ID NO: 3],TATGAGTGCTCCTGGCAGTAT; P2 [SEQ ID NO: 4], GCCATGCTCCAATCACTCCAG; P3 [SEQID NO: 5], AATGTGCTCGCCAAAACTCG; P4 [SEQ ID NO: 6],CGCAGTCTTATGGGTCCTCC; P5 [SEQ ID NO: 7], CTGCCTCTGCCTCTGAGTCT; P6 [SEQID NO: 8], GCCAATGTATCGAGACTGC. IL12Rβ1 transcripts were amplified byPCR in a 25-μl reaction comprising the following: 2.5 uL of a 10×PCRbuffer (200 mM Tris pH 8.4, 500 mM KCl), 0.5 uL of 10 mM dNTPs, 1 uL 50mM MgCl₂, 0.1 uL of 5 U/uL Taq polymerase (Invitrogen), 1 uL of 5 μMforward primer (P1 or P3), 1 uL of 5 uM reverse primer (P2, P4, P5 orP6), 17.9 uL of DNAse-free H₂O and 1 uL of cDNA (a minimum of 200 pgcDNA). Following denaturation at 94° C. for 3 min, the reaction wascycled forty times under the following conditions: 94° C. for 45seconds, 55° C. for 30 sec, 72° C. for 90 sec. The products of thisreaction were either analyzed on a 2% agarose gel or kept for IL12Rβ1Spectratype analysis as described below.

IL12Rβ1-Spectratype Analysis

IL12Rβ1-Spectratype analysis of IL12Rβ1 and IL12Rβ1ΔTM mRNAs—and thequantification of the resultant data—was a modification of the nowcommonly used TCR-CD3 Spectratype analysis (Pannetier et al., Proc NatlAcad Sci USA, 90:4319-4323, 1993). IL12Rβ1 and IL12Rβ1ΔTM cDNAs werefirst amplified by PCR in the 25-μl reaction detailed above with theforward primer 5′-GCAGCCGAGTGATGTACAAG-3′ [SEQ ID NO: 9] and reverseprimer 5′-CTGCCTCTGCCTCTGAGTCT-3′ [SEQ ID NO: 7]. The forward primercorresponds to nucleotides 1653-1672 of the mouse IL12Rβ1 transcript andprecedes the transmembrane-encoding sequence (nucleotides 1739-1834).The reverse primer is downstream of the transmembrane-encoding sequence,corresponding to nucleotides 2067-2086 of the mouse IL12Rβ1 transcript.To fluorescently label the IL12Rβ1 and IL12Rβ1ΔTM amplicons a secondrunoff PCR reaction was performed as follows: 2.5 μl of the initialamplification reaction was added to 22.5 uL of a second PCR comprising2.5 uL of 10×PCR buffer, 0.5 uL of 10 mM dNTPs, 1 uL 50 mM MgCl₂, 0.1 uLof 5 U/uL Taq polymerase (Invitrogen), 2.0 uL of a 5 uM FAM-labeledreverse primer (FAM-5′-AGTGCTGCCACAGGGTGTA-3′ [SEQ ID NO: 10]), and 16.4uL of DNAse-free H₂O (final volume: 25 uL). Following denaturation at94° C. for 5 min, the reaction was cycled four times under the followingconditions: 95° C. for 2 minutes, 55° C. for 2 minutes, 72° C. for 20minutes. 2.0 μL of the completed runoff PCR reaction was then added to2.0 μl of ROX-500 size standard (Applied Biosystems) and 36 μl of HiDiFormamide (Applied Biosystems). Following denaturation, the productswere detected and their size and relative amount determined using anApplied Biosystems 3100 sequencer analyzed with GeneScan software(Applied Biosystems). For calculating the ratio of IL12Rβ1ΔTM to IL12Rβ1(i.e. IL12Rβ1ΔTM:IL12Rβ1) the area under the IL12Rβ1ΔTM peak was dividedby the area under the reference IL12Rβ1 peak.

Plasmids and Transfections

Plasmids expressing IL12Rβ1 and IL12Rβ1ΔTM cDNAs in vector pEF-BOS(Mizushima and Nagata, Nucleic Acids Res, 18:5322, 1990) under the EF1αpromoter have been described (Chua et al., J Immunology 155:4286-4299,1995) (pEF-BOS.IL12Rβ1 and pEF-BOS.IL12Rβ1ΔTM). pAcGFP1-N1 (ClontechLaboratories, Mountain View, Calif.) was used to express eGFP under theCMV promoter to identify transfected cells. For transfection intoNIH/3T3 cells (ATCC, Manassas, Va.) the Polyfect system (Qiagen,Valencia, Calif.) was used as per the manufacturers instructions.

Western Blot Analysis

SDS-PAGE analysis of reduced protein samples and subsequent transfer toPVDF membrane was performed using standard protocols. Membranes weresubsequently probed overnight with 400 ng/mL goat polyclonalanti-IL12Rβ1 (R&D Systems) in a solution of Tris-buffered saline (TBS)containing 2.5% powdered milk, washed with TBS, secondarily probed withHRP-conjugated anti-goat IgG and detected using ECL western blottingsubstrate (ThermoScientific, Rockford, Ill.) for chemiluminescence. Fora positive control, recombinant mouse IL12Rβ1 (R&D Systems) was runsimultaneously with each gel.

Determination of Total NFκB and Phospho-NFκB Levels

C57BL/6 and IL12Rβ1^(−/−) BMDCs were exposed to M. tuberculosis or mediaalone for indicated times. Following each time point, cells werecollected and washed with ice-cold PBS. Cells were subsequently lysed byaddition of ice-cold lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1 mMEDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM Na₄P₂O₇, 1 mMβ-glycerophosphate, 1 mM Na₃VO₄, 1 ug/mL leupeptin plus 1 mM PMSF) andsonicated on ice. Total lysates were centrifuged at 14000 RPM for 10minutes at 4° C.; the supernatants were aliquoted and stored at −80° C.until determination of total NFκB and phospho-NFκB levels by ELISA(PathScan Inflammation Multi-Target Sandwich ELISA; Cell SignalingTechnology, Danvers, Mass.).

NFκB Electromobility Shift Assay (EMSA)

Nuclear extracts from indicated cell populations were subjected topolyacrylamide electrophoresis and EMSA analysis of subsequentlygenerated blots with Panomics NFκB EMSA Kit (Fremont, Calif.) withbiotinylated NFκB probe 5′-AGTTGAGGGGACTTTCCCAGGC-3′ [SEQ ID NO: 11] asper the manufacturers' instructions.

In Vitro mRNA Transcription

To generate in vitro transcribed (IVT) mRNA of IL12Rβ1, IL12Rβ1ΔTM andeGFP it was first necessary to subclone their respective cDNAs into asecond plasmid downstream of a T7 phage polymerase. The IL12Rβ1 andIL12Rβ1ΔTM cDNAs were first amplified out of their pEF-BOS backbonesusing primers that flanked their start and stop codons; specifically5′-TGTTTCTGAGCGTGGACAAG-3′ [SEQ ID NO: 12] and 5′-CCGCAGTCTTATGGGTCCT-3′[SEQ ID NO: 13]. eGFP was amplified out of pAcGFP1-N1 using primers5′-TAGCGCTACCGGACTCAGAT-3′ [SEQ ID NO: 14] (cognate to the sequence just5′ of the eGFP start codon) and 5′-GGGAGGTGTGGGAGGTTTT-3′ [SEQ ID NO:15]. IL12Rβ1, IL12Rβ1ΔTM and eGFP amplicons were subsequently TA-clonedinto pCR2.1 downstream of the T7 phage polymerase promoter to generatethe plasmids pCR2.1.IL12Rβ1, pCR2.1.IL12Rβ1ΔTM and pCR2.1.eGFP,respectively. These constructs were subsequently used in the mMessagemMachine kit (Ambion) to generate 5′ capped IVT mRNA as per themanufacturers instruction. mRNA quality was checked by gelelectrophoresis and the concentration determined by spectrophotometricanalysis at OD₂₆₀. mRNA aliquots were stored at −80° C. until use fortransfections.

Electroporation of DCs

Electroporation of individual mRNAs into IL12Rβ1^(−/−) DCs was done asperformed by Ponsaerts et al. (Ponsaerts et al., Leukemia, 16,1324-1330, 2002) with minor modifications. Briefly, prior toelectroporation, DCs were washed twice with electroporation buffer(Ambion) and resuspended to a final concentration of 5×10⁷ cells/ml inelectroporation buffer. 0.2 ml of the cell suspension was then mixedwith 20 μg of IVT mRNA and electroporated in a 0.4 cm cuvette at 300 Vand 150 μF using a Gene Pulser Xcell Electroporation System (BioRad).After electroporation, fresh complete medium was added to the cellsuspension followed by incubation at 37° C. in a humidified atmospheresupplemented with 5% CO₂. For all electroporation experiments theco-transfection of eGFP-mRNA was used to both confirm transfectionefficiency and to identify cells that were successfully transfected.

In Vivo Migration of Electroporated DCs

Following mRNA electroporation and overnight culture, 1×10⁶ DCs werecultured with 10 μg/mL irradiated M. tuberculosis and 1 μM ESAT₁₋₂₀peptide for 3 hrs. DCs were then washed, resuspended in PBS andinstilled via the trachea into the lungs of Thy1.1 congenic mice.Eighteen hours prior to instillation each mouse had intravenouslyreceived 5×10⁶ CFSE-labeled ESAT-TCR CD4⁺ cells. The surface expressionof CD44 and CD69 on CFSE⁺CD4⁺ cells in the draining MLNs was assessed 12hours later by flow cytometry.

IL12Rβ1 Isoform Expression by Human DCs

Monocyte-derived DCs were generated by incubating CD14+ monocytes(magnetically purified from apheresis samples) with GMCSF (20 ng/ml,Peprotech) and IL4 (50 ng/ml, R&D) for 7 days. DCs were then incubatedfor 24 h with LPS (1 ?g/ml) or for 3 days with either of the following:IL1? (10 ng/ml), IL10 (200 ng/ml), IL6 (10 ng/ml), IL2 (20 U/ml), CCL3(50 ng/ml), P1GF (50 ng/ml) or RPMI media (control). Alternatively, DCswere stimulated with M. tuberculosis over a 6-hour period. cDNAgenerated from these populations was then amplified with primer pairsthat either amplified both IL12Rβ1 isoforms 1 and 2 (Common;5′-ACACTCTGGGTGGAATCCTG-3′ [Forward][SEQ ID NO: 1] and5′GCCAACTTGGACACCTTGAT-3′ [Reverse][SEQ ID NO: 2]), only isoform 1(Isoform 1 Specific; 5′-ACACTCTGGGTGGAATCCTG-3′ [Forward][SEQ ID NO: 1]and 5′CACCCTCTCTGAGCCTCAAC-3′ [Reverse][SEQ ID NO: 16]) or only isoform2 (Isoform 2 Specific; 5′-ACACTCTGGGTGGAATCCTG-3′ [Forward][SEQ ID NO:1] and 5′CTAGCACTTTGGGAGGTGGA-3′ [Reverse][SEQ ID NO: 17]). Theconditions used to amplify with these primers were the same as thoseused for the primary PCR of IL12Rβ1 Spectratype analysis detailed above.cDNA from CD3+ PBMCs was used as a positive control for IL12Rβ1expression. Amplicons were analyzed by 2% agarose gel electrophoresis.

Statistical Analysis

Differences between the means of experimental groups were analyzed withthe two-tailed Student's t-test as the data was considered parametric.Differences with a P value of 0.05 or less were considered significant.Prism software was used for all analyses.

We claim:
 1. A method of identifying an active M. tuberculosisinfection, comprising: a) providing a patient infected with M.tuberculosis, b) determining the amount of IL12Rβ1 isoform 1 mRNA andIL12Rβ1 isoform 2 mRNA in a cell from said patient, wherein said cell isa peripheral blood mononuclear cell, c) diagnosing the patient as havingan active M. tuberculosis infection if the amount of IL12Rβ1 isoform 2mRNA is greater than the amount of IL12Rβ1 isoform 1 mRNA, d) initiatinga therapeutic treatment on said patient with an active M. tuberculosisinfection.
 2. The method of claim 1, wherein said cell is a peripheralblood mononuclear cell.
 3. The method of claim 1, wherein the amount ofIL12Rβ1 isoform 1 mRNA and IL12Rβ1 isoform 2 mRNA is determined by PCR.4. The method of claim 3, wherein said PCR is real-time PCR.
 5. Themethod of claim 1, wherein the amount of IL12Rβ1 isoform 1 mRNA andIL12Rβ1 isoform 2 mRNA is determined by microarray transcriptionalanalysis.
 6. A method of identifying an active M. tuberculosisinfection, comprising: a) providing a patient suspected of beinginfected with M. tuberculosis, b) determining the amount of IL12Rβ1isoform 1 mRNA and IL12Rβ1 isoform 2 mRNA in a cell from said patient,wherein said cell is a peripheral blood mononuclear cell, c) diagnosingthe patient as having an active M. tuberculosis infection if the amountof IL12Rβ1 isoform 2 mRNA is greater than the amount of IL12Rβ1 isoform1 mRNA, d) initiating a therapeutic treatment on said patient with anactive M. tuberculosis infection.
 7. A method of monitoring a M.tuberculosis infection, comprising: a) providing a patient infected withM. tuberculosis, b) determining the amount of IL12Rβ1 isoform 1 mRNA andIL12Rβ1 isoform 2 mRNA in a cell from said patient, wherein said cell isa peripheral blood mononuclear cell, c) diagnosing the patient as havingan active M. tuberculosis infection if the amount of IL12Rβ1 isoform 2mRNA is greater than the amount of IL12Rβ1 isoform 1 mRNA, d) diagnosingthe patient as having a latent M. tuberculosis infection if the amountof IL12Rβ1 isoform 1 mRNA is greater than the amount of IL12Rβ1 isoform2 mRNA, c) initiating a therapeutic treatment if the patient has anactive M. tuberculosis infection.
 8. The method of claim 7, wherein saidcell is a peripheral blood mononuclear cell.
 9. The method of claim 7,wherein the amount of IL12Rβ1 isoform 1 mRNA and IL12Rβ1 isoform 2 mRNAis determined by PCR.
 10. The method of claim 9, wherein said PCR isreal-time PCR.
 11. The method of claim 7, wherein the amount of IL12Rβ1isoform 1 mRNA and IL12Rβ1 isoform 2 mRNA is determined by microarraytranscriptional analysis.
 12. A method of monitoring a patient'sresponse to treatment for an active M. tuberculosis infection,comprising: a) providing a patient with an active M. tuberculosisinfection, b) initiating a therapeutic treatment on said patient, b)determining the amount of IL12Rβ1 isoform 1 mRNA and IL12Rβ1 isoform 2mRNA in a cell from said patient, wherein said cell is a peripheralblood mononuclear cell, and c) continuing said therapeutic treatment ifthe ratio of IL12Rβ1 isoform 1 mRNA to IL12Rβ1 isoform 2 mRNA increases.13. The method of claim 12, wherein said therapeutic treatment isdetermined to be effective if the ratio of IL12Rβ1 isoform 2 mRNA toIL12Rβ1 isoform 1 mRNA decreases.
 14. The method of claim 12, whereinsaid cell is a peripheral blood mononuclear cell.
 15. The method ofclaim 12, wherein the amount of IL12Rβ1 isoform 1mRNA and IL12Rβ1isoform 2 mRNA is determined by PCR.
 16. The method of claim 15 whereinsaid PCR is real-time PCR.
 17. The method of claim 12, wherein theamount of IL12Rβ1 isoform 1 mRNA and IL12Rβ1 isoform 2 mRNA isdetermined by microarray transcriptional analysis.
 18. A method ofmonitoring a patient's response to treatment for an active M.tuberculosis infection, comprising: a) providing a patient with anactive M. tuberculosis infection, b) initiating a therapeutic treatmenton said patient, b) determining the amount of IL12Rβ1 isoform 1 mRNA andIL12Rβ1 isoform 2 mRNA in a cell from said patient during the course ofsaid treatment, wherein said cell is a peripheral blood mononuclear, andc) diagnosing said patient as responding to said treatment when theratio of IL12Rβ1 isoform 2 mRNA to IL12Rβ1 isoform 1 mRNA decreases. 19.The method of claim 18, wherein said cell is a peripheral bloodmononuclear cell.
 20. The method of claim 18, wherein the amount ofIL12Rβ1 isoform 1mRNA and IL12Rβ1 isoform 2 mRNA is determined by PCR.21. The method of claim 20 wherein said PCR is real-time PCR.
 22. Themethod of claim 18, wherein the amount of IL12Rβ1 isoform 1 mRNA andIL12Rβ1 isoform 2 mRNA is determined by microarray transcriptionalanalysis.